Nucleic acid ligand complexes

ABSTRACT

This invention discloses a method for preparing a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipophilic compound or non-immunogenic, high molecular weight compound by identifying a nucleic acid ligand by SELEX methodology and associating the nucleic acid ligand with a lipophilic compound or a non-immunogenic, high molecular weight compound. The invention further discloses complexes comprising one or more nucleic acid ligands in association with a lipophilic compound or non-immunogenic, high molecular weight compound.

FIELD OF THE INVENTION

This invention relates to a method for preparing a therapeutic ordiagnostic Complex comprised of a Nucleic Acid Ligand and a LipophilicCompound or Non-Immunogenic, High Molecular Weight Compound byidentifying a Nucleic Acid Ligand by SELEX methodology and associatingthe Nucleic Acid Ligand with a Lipophilic Compound or a Non-Immunogenic,High Molecular Weight Compound. The invention further relates toimproving the pharmacokinetic properties of a Nucleic Acid Ligand byassociating the Nucleic Acid Ligand to a Lipophilic Compound orNon-Immunogenic, High Molecular Weight Compound to form a Complex. Theinvention further relates to a method for targeting a therapeutic ordiagnostic agent to a specific predetermined biological Target byassociating the agent with a Complex comprised of a Nucleic Acid Ligandand a Lipophilic Compound or Non-Immunogenic, High Molecular WeightCompound, wherein the Nucleic Acid Ligand has a SELEX Target associatedwith the specific predetermined Target and the Nucleic Acid Ligand isassociated with the exterior of the Complex. The invention also includescomplexes comprising one or more Nucleic Acid Ligand in association witha Lipophilic Compound or Non-Immunogenic, High Molecular WeightCompound.

BACKGROUND OF THE INVENTION

A. Selex

The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by EXponential enrichment, termed SELEX, it has become clearthat nucleic acids have three dimensional structural diversity notunlike proteins. SELEX is a method for the in vitro evolution of nucleicacid molecules with highly specific binding to target molecules and isdescribed in U.S. patent application Ser. No. 07/536,428, filed Jun. 11,1990, entitled “Systematic Evolution of Ligands by ExponentialEnrichment”, now abandoned, U.S. patent application Ser. No. 07/714,131,filed Jun. 10, 1991, entitled “Nucleic Acid Ligands”, now U.S. Pat. No.5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17,1992, entitled “Nucleic Acid Ligands”, now U.S. Pat. No. 5,270.163 (seealso PCT/US91/04078), each of which is specifically incorporated byreference herein. Each of these applications, collectively referred toherein as the SELEX Patent Applications, describes a fundamentally novelmethod for making a Nucleic Acid Ligand to any desired target molecule.The SELEX process provides a class of products which are referred to asNucleic Acid Ligands, each ligand having a unique sequence, and whichhas the property of binding specifically to a desired target compound ormolecule. Each SELEX-identified Nucleic Acid Ligand is a specific ligandof a given target compound or molecule. SELEX is based on the uniqueinsight that Nucleic Acids have sufficient capacity for forming avariety of two- and three-dimensional structures and sufficient chemicalversatility available within their monomers to act as ligands (formspecific binding pairs) with virtually any chemical compound, whethermonomeric or polymeric. Molecules of any size or composition can serveas targets.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of Nucleic Acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound Nucleic Acids from those Nucleic Acidswhich have bound specifically to target molecules, dissociating theNucleic Acid-target complexes, amplifying the Nucleic Acids dissociatedfrom the Nucleic Acid-target complexes to yield a ligand-enrichedmixture of Nucleic Acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific high affinity Nucleic Acid Ligands tothe target molecule.

It has been recognized by the present inventors that the SELEX methoddemonstrates that Nucleic Acids as chemical compounds can form a widearray of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby Nucleic Acids in biological systems.

The present inventors have recognized that SELEX or SELEX-like processescould be used to identify Nucleic Acids which can facilitate any chosenreaction in a manner similar to that in which Nucleic Acid Ligands canbe identified for any given target. In theory, within a CandidateMixture of approximately 10¹³ to 10¹⁸ Nucleic Acids, the presentinventors postulate that at least one Nucleic Acid exists with theappropriate shape to facilitate each of a broad variety of physical andchemical interactions.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure.” describes the use of SELEX in conjunction with gelelectrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” describes a SELEX based method for selectingNucleic Acid Ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine,” describes a method for identifying highlyspecific Nucleic Acid Ligands able to discriminate between closelyrelated molecules, which can be non-peptidic, termed Counter-SELEX. U.S.patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled“Systematic Evolution of Ligands by EXponential Enrichment: SolutionSELEX,” describes a SELEX-based method which achieves highly efficientpartitioning between oligonucleotides having high and low affinity for atarget molecule.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides,” that describesoligonucleotides containing nucleotide derivatives chemically modifiedat the 5- and 2′-positions of pyrimidines. U.S. patent application Ser.No. 08/134,028, supra, describes highly specific Nucleic Acid Ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of 2′ Modified Pyrimidine Intramolecular NucleophilicDisplacement”, describes oligonucleotides containing various 2′-modifiedpyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284.063, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX” and U.S. patent application Ser. No.08/234,997, filed Apr. 28, 1994, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Blended SELEX,” respectively. Theseapplications allow the combination of the broad array of shapes andother properties, and the efficient amplification and replicationproperties, of oligonucleotides with the desirable properties of othermolecules. Each of the above described patent applications whichdescribe modifications of the basic SELEX procedure are specificallyincorporated by reference herein in their entirety.

B. Lipid Constructs

Lipid Bilayer Vesicles are closed, fluid-filled microscopic sphereswhich are formed principally from individual molecules having polar(hydrophilic) and non-polar (lipophilic) portions. The hydrophilicportions may comprise phosphato, glycerylphosphato, carboxy, sulfato,amino, hydroxy, choline or other polar groups. Examples of lipophilicgroups are saturated or unsaturated hydrocarbons such as alkyl, alkenylor other lipid groups. Sterols (e.g., cholesterol) and otherpharmaceutically acceptable adjuvants (including anti-oxidants likealpha-tocopherol) may also be included to improve vesicle stability orconfer other desirable characteristics.

Liposomes are a subset of these bilayer vesicles and are comprisedprincipally of phospholipid molecules that contain two hydrophobic tailsconsisting of fatty acid chains. Upon exposure to water, these moleculesspontaneously align to form spherical, bilayer membranes with thelipophilic ends of the molecules in each layer associated in the centerof the membrane and the opposing polar ends forming the respective innerand outer surface of the bilayer membrane(s). Thus, each side of themembrane presents a hydrophilic surface while the interior of themembrane comprises a lipophilic medium. These membranes may be arrangedin a series of concentric, spherical membranes separated by thin strataof water, in a manner not dissimilar to the layers of an onion, aroundan internal aqueous space. These multilamellar vesicles (MLV) can beconverted into small or Unilamellar Vesicles (UV), with the applicationof a shearing force.

The therapeutic use of liposomes includes the delivery of drugs whichare normally toxic in the free form. In the liposomal form, the toxicdrug is occluded, and may be directed away from the tissues sensitive tothe drug and targeted to selected areas. Liposomes can also be usedtherapeutically to release drugs over a prolonged period of time,reducing the frequency of administration. In addition, liposomes canprovide a method for forming aqueous dispersions of hydrophobic oramphiphilic drugs, which are normally unsuitable for intravenousdelivery.

In order for many drugs and imaging agents to have therapeutic ordiagnostic potential, it is necessary for them to be delivered to theproper location in the body, and the liposome can thus be readilyinjected and form the basis for sustained release and drug delivery tospecific cell types, or parts of the body. Several techniques can beemployed to use liposomes to target encapsulated drugs to selected hosttissues, and away from sensitive tissues. These techniques includemanipulating the size of the liposomes, their net surface charge, andtheir route of administration. MLVs, primarily because they arerelatively large, are usually rapidly taken up by thereticuloendothelial system (principally the liver and spleen). UVs, onthe other hand, have been found to exhibit increased circulation times,decreased clearance rates and greater biodistribution relative to MLVs.

Passive delivery of liposomes involves the use of various routes ofadministration, e.g., intravenous, subcutaneous, intramuscular andtopical. Each route produces differences in localization of theliposomes. Two common methods used to direct liposomes actively toselected target areas involve attachment of either antibodies orspecific receptor ligands to the surface of the liposomes. Antibodiesare known to have a high specificity for their corresponding antigen andhave been attached to the surface of liposomes, but the results havebeen less than successful in many instances. Some efforts, however, havebeen successful in targeting liposomes to tumors without the use ofantibodies, see, for example, U.S. Pat. No. 5,019,369.

An area of development aggressively pursued by researchers is thedelivery of agents not only to a specific cell type but into the cell'scytoplasm and, further yet, into the nucleus. This is particularlyimportant for the delivery of biological agents such as DNA, RNA,ribozymes and proteins. A promising therapeutic pursuit in this areainvolves the use of antisense DNA and RNA oligonucleotides for thetreatment of disease. However, one major problem encountered in theeffective application of antisense technology is that oligonucleotidesin their phosphodiester form are quickly degraded in body fluids and byintracellular and extracellular enzymes, such as endonucleases andexonucleases, before the target cell is reached. Intravenousadministration also results in rapid clearance from the bloodstream bythe kidney, and uptake is insufficient to produce an effectiveintracellular drug concentration. Liposome encapsulation protects theoligonucleotides from the degradative enzymes, increases the circulationhalf-life and increases uptake efficiency as a result of phagocytosis ofthe Liposomes. In this way, oligonucleotides are able to reach theirdesired target and to be delivered to cells in vivo.

A few instances have been reported where researchers have attachedantisense oligonucleotides to Lipophilic Compounds or Non-Immunogenic,High Molecular Weight Compounds. Antisense oligonucleotides, however,are only effective as intracellular agents. Antisenseoligodeoxyribonucleotides targeted to the epidermal growth factor (EGF)receptor have been encapsulated into Liposomes linked to folate via apolyethylene glycol spacer (folate-PEG-Liposomes) and delivered intocultured KB cells via folate receptor-mediated endocytosis (Wang el al.(1995) 92:3318-3322). In addition, a Lipophilic Compound covalentlyattached to an antisense oligonucleotide has been demonstrated in theliterature (EP 462 145 B1).

SUMMARY OF THE INVENTION

The present invention provides a method for preparing a therapeutic ordiagnostic Complex comprised of a Nucleic Acid Ligand and a LipophilicCompound or Non-Immunogenic, High Molecular Weight Compound by themethod comprising identifying a Nucleic Acid Ligand from a CandidateMixture of Nucleic Acids where the Nucleic Acid is a ligand of a giventarget by the method of (a) contacting the Candidate Mixture of NucleicAcids with the target, (b) partitioning between members of saidCandidate Mixture on the basis of affinity to the target, and c)amplifying the selected molecules to yield a mixture of Nucleic Acidsenriched for Nucleic Acid sequences with a relatively higher affinityfor binding to the target, and associating said identified Nucleic AcidLigand with a Lipophilic Compound or a Non-Immunogenic, High MolecularWeight Compound.

In another embodiment, this invention provides a method for improvingthe cellular uptake of a Nucleic Acid Ligand having an intracellularSELEX Target by associating the Nucleic Acid Ligand with a LipophilicCompound or a Non-Immunogenic, High Molecular Weight Compound to form aComplex comprised of a Nucleic Acid Ligand and a Lipophilic Compound ora Non-Immunogenic, High Molecular Weight Compound and administering theComplex to a patient.

In another embodiment, this invention provides a method for improvingthe pharmacokinetic properties of a Nucleic Acid Ligand by associatingthe Nucleic Acid Ligand to a Lipophilic Compound or a Non-Immunogenic,High Molecular Weight Compound to form a Complex comprised of a NucleicAcid Ligand and a Lipophilic Compound or a Non-Immunogenic, HighMolecular Weight Compound and administering the Complex to a patient.

In another embodiment, this invention provides a method for targeting atherapeutic or diagnostic agent to a specific predetermined biologicalTarget in a patient comprising associating the therapeutic or diagnosticagent with a Complex comprised of a Nucleic Acid Ligand and a LipophilicCompound or Non-Immunogenic, High Molecular Weight Compound, wherein theNucleic Acid Ligand has a SELEX Target associated with the specificpredetermined Target, and the Nucleic Acid Ligand is associated with theexterior of the Complex and administering the Complex to a patient.

It is an object of the present invention to provide Complexes comprisingone or more Nucleic Acid Ligands in association with a LipophilicCompound or Non-Immunogenic, High Molecular Weight Compound and methodsfor producing the same. It is a further object of the invention toprovide one or more Nucleic Acid Ligands in association with aLipophilic Compound or Non-Immunogenic, High Molecular Weight Compoundwith Improved Pharmacokinetic Properties. In another aspect of theinvention, the Lipophilic Compound is a Lipid Construct. In thisembodiment, the Lipid Construct is preferably a Lipid Bilayer Vesicleand most preferably a Liposome. In certain embodiments of the inventionthe Lipophilic Compound is cholesterol, dialkyl glycerol, or diacylglycerol. In another embodiment of the invention, the Non-Immunogenic,High Molecular Weight Compound is PEG. In another embodiment of theinvention, the Non-Immunogenic, High Molecular Weight Compound ismagnetite. In the preferred embodiment, the Nucleic Acid Ligand isidentified according to the SELEX method.

In embodiments of the invention directed to Complexes comprisingcholesterol, dialkyl glycerol, diacyl glycerol, PEG, or magnetite inassociation with a Nucleic Acid Ligand or ligands, the Nucleic AcidLigand or ligands can serve in a targeting capacity.

In embodiments of the invention directed to Complexes comprising a LipidConstruct where the Lipid Construct is of a type that has a membranedefining an interior compartment such as a Lipid Bilayer Vesicle, theNucleic Acid Ligand in association with the Lipid Construct may beassociated with the membrane of the Lipid Construct or encapsulatedwithin the compartment. In embodiments where the Nucleic Acid Ligand isin association with the membrane. the Nucleic Acid Ligand can associatewith the interior-facing or exterior-facing part of the membrane, suchthat the Nucleic Acid Ligand is projecting in to or out of the vesicle.In embodiments where the Nucleic Acid Ligand is projecting out of theComplex, the Nucleic Acid Ligand can serve in a targeting capacity.Non-Immunogenic, High Molecular Weight Compounds can also be associatedwith the membrane. In one embodiment, the Nucleic Acid Ligand may beassociated with a Non-Immunogenic, High Molecular Weight Compound whichis associated with the membrane. The membrane may have associated withit additional Non-Immunogenic, High Molecular Weight Compounds notassociated with a Nucleic Acid Ligand.

In embodiments where the Nucleic Acid Ligand of the Complex serves in atargeting capacity, the Complex can incorporate or have associated withit therapeutic or diagnostic agents. In one embodiment, the therapeuticagent is a drug. In an alternative embodiment, the therapeutic ordiagnostic agent is one or more additional Nucleic Acid Ligands. NucleicAcid Ligands specific for different targets can project from theexternal surface of the Complex. The Complex can project from theexternal surface one or more Nucleic Acid Ligands which are specific fordifferent SELEX Targets on the same Target.

These and other objects, as well as the nature, scope and utilization ofthis invention, will become readily apparent to those skilled in the artfrom the following description and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1Y show the molecular descriptions of NX229, NX232, NX253,NX256, 225T3, 225T3N, T-P4, NX-256-PEG-20,000, 225T3N-PEG-3400,T-P4-PEG-(20,000 or 10,000), NX268, NX191, JW966, NX278, JW986, NX213,NX244, JW1130, NX287, JW1336-20K PEG, JW1379, JW1380, scNX278,JW986-PEG-(10,000, 20,000, or 40,000), and JW1336 (SEQ ID NOS:6-30). Alower case letter preceding a nucleotide indicates the following:m=2′-O-Methyl, a=2′-amino, r=ribo, and f=2′-fluoro. No lener preceding anucleotide indicates a deoxyribonucleotide (2′H). An S following anucleotide denotes a backbone modification consisting of aphosphorothioate internucleoside linkage.

FIG. 2 shows gel permeation chromatograms for empty Liposomes (Empty),Liposomes with 4.7 mg NX232 (L-NeX2a), Liposomes with 11.8 mg NX232(L-NeX2b), free NX232 at 72 mg (Free 72 mg), free NX232 at 7.2 mg (Free7.2 mg), and free NX232 at 10 mg which had been sonicated (Free/soni 10mg).

FIG. 3 summarizes the data for the plasma concentration of NX229, NX232,NX253, NX253 +Liposome, and NX256-PEG20K as a function of time followingthe bolus injection.

FIG. 4 summarizes the data for the plasma concentration of NX213 (SEQ IDNO:21), NX268 (SEQ ID NO: 16), NX278 (SEQ ID NO: 19), NX278+liposome,JW986 (SEQ ID NO:20), NX213 liposome encapsulated, and NX244 (SEQ IDNO:22) as a function of time following the bolus injection.

FIG. 5 summarizes the data for the plasma concentration of JW966 (SEQ IDNO: 18) and JW966+liposome as a function of time following the bolusinjection.

FIG. 6 summarizes the data for the plasma concentration of NX268 (SEQ IDNO: 16) and NX268+liposome as a function of time following the bolusinjection.

FIG. 7 summarizes the data for the plasma concentration of NX191 (SEQ IDNO:17), JW986+PEG20K, PEG40K, and PEG10K (SEQ ID NO:29) as a function oftime following the bolus injection.

FIG. 8 summarizes the data for the plasma concentration of JW986+PEG20K(SEQ ID NO:29) and JW1 130 (SEQ ID NO:23) as a function of timefollowing the bolus injection.

FIG. 9 summarizes the data for the plasma concentration of NX287+PEG40K(SEQ ID NO:24) and NX256 (SEQ ID NO:9) as a function of time followingthe bolus injection.

FIG. 10 summarizes the data for the plasma concentration of JW1130 (SEQID NO:23), 1136-PEG20K (SEQ ID NO:25), 1336 (SEQ ID NO:30), and 1379/80(SEQ ID NOS:26-27) as a function of time following the bolus injection.

FIG. 11 shows the chromatograms for NX232 (SEQ ID NO:7), NX232+1% PGSUV,NX232+2.5% PGSUV, and NX232+PGSUV.

FIG. 12 shows the fraction of bound Nucleic Acid Ligand (NX253) (SEQ IDNO:8) as a function of Liposome:Nucleic Acid Ligand ratio.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

“Covalent Bond” is the chemical bond formed by the sharing of electrons.

“Non-Covalent Interactions” are means of holding together molecularentities by interactions other than Covalent Bonds including ionicinteractions and hydrogen bonds.

“Lipid Constructs,” for purposes of this invention, are structurescontaining lipids, phospholipids, or derivatives thereof comprising avariety of different structural arrangements which lipids are known toadopt in aqueous suspension. These structures include, but are notlimited to, Lipid Bilayer Vesicles, micelles, Liposomes, emulsions,lipid ribbons or sheets, and may be complexed with a variety of drugsand adjuvants which are known to be pharmaceutically acceptable. Commonadjuvants include cholesterol and alpha-tocopherol, among others. TheLipid Constructs may be used alone or in any combination which oneskilled in the art would appreciate to provide the characteristicsdesired for a particular application. In addition, the technical aspectsof Lipid Constructs and Liposome formation are well known in the art andany of the methods commonly practiced in the field may be used for thepresent invention.

“Lipophilic Compounds” are compounds which have the propensity toassociate with or partition into lipid and/or other materials or phaseswith low dielectric constants, including structures that are comprisedsubstantially of lipophilic components. Lipophilic Compounds includeLipid Constructs as well as non-lipid containing compounds that have thepropensity to associate with lipid (and/or other materials or phaseswith low dielectric constants). Cholesterol, phospholipid, and dialkylglycerol are further examples of Lipophilic Compounds.

“Complex” as used herein describes the molecular entity formed by theassociation of a Nucleic Acid Ligand with a Lipophilic Compound orNon-Immunogenic, High Molecular Weight Compound. The association can bethrough either Covalent Bonds or Non-Covalent Interactions.

“Nucleic Acid Ligand” as used herein is a non-naturally occurringNucleic Acid having a desirable action on a SELEX Target. A desirableaction includes, but is not limited to, binding of the SELEX Target,catalytically changing the SELEX Target, reacting with the SELEX Targetin a way which modifies/alters the SELEX Target or the functionalactivity of the SELEX Target, covalently attaching to the SELEX Targetas in a suicide inhibitor, facilitating the reaction between the Targetand another molecule. In the preferred embodiment, the action isspecific binding affinity for a Target molecule. such Target moleculebeing a three dimensional chemical structure other than a polynucleotidethat binds to the Nucleic Acid Ligand through a mechanism whichpredominantly depends on Watson/Crick base pairing or triple helixbinding, wherein the Nucleic Acid Ligand is not a Nucleic Acid havingthe known physiological function of being bound by the Target molecule.In preferred embodiments of the invention, the Nucleic Acid Ligand ofthe Complexes of the invention are identified by the SELEX methodology.Nucleic Acid Ligands include Nucleic Acids that are identified from aCandidate Mixture of Nucleic Acids, said Nucleic Acid being a ligand ofa given Target, by the method comprising a) contacting the CandidateMixture with the Target, wherein Nucleic Acids having an increasedaffinity to the Target relative to the Candidate Mixture may bepartitioned from the remainder of the Candidate Mixture; b) partitioningthe increased affinity Nucleic Acids from the remainder of the CandidateMixture; and c) amplifying the increased affinity Nucleic Acids to yielda ligand-enriched mixture of Nucleic Acids.

“Candidate Mixture” is a mixture of Nucleic Acids of differing sequencefrom which to select a desired ligand. The source of a Candidate Mixturecan be from naturally-occurring Nucleic Acids or fragments thereof,chemically synthesized Nucleic Acids, enzymatically synthesized NucleicAcids or Nucleic Acids made by a combination of the foregoingtechniques. In a preferred embodiment, each Nucleic Acid has fixedsequences surrounding a randomized region to facilitate theamplification process.

“Nucleic Acid” means either DNA, RNA, single-stranded or double-strandedand any chemical modifications thereof. Modifications include, but arenot limited to, those which provide other chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction, and fluxionality to the Nucleic Acid Ligandbases or to the Nucleic Acid Ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil, backbone modifications such asinternucleoside phosphorothioate linkages, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping.

“Non-Immunogenic, High Molecular Weight Compound” is a compound ofapproximately 1000 Da or more that typically does not generate animmunogenic response. For the purposes of this invention, an immunogenicresponse is one that causes the organism to make antibody proteins.Examples of Non-Immunogenic, High Molecular Weight Compounds includepolyethylene glycol (PEG); polysaccharides, such as dextran;polypeptides, such as albumin; and magnetic structures; such asmagnetite. In certain embodiments, the Non-Immunogenic, High MolecularWeight Compound can also be a Nucleic Acid Ligand.

“Lipid Bilayer Vesicles” are closed, fluid-filled microscopic sphereswhich are formed principally from individual molecules having polar(hydrophilic) and non-polar (lipophilic) portions. The hydrophilicportions may comprise phosphato, glycerylphosphato, carboxy, sulfato,amino, hydroxy, choline and other polar groups. Examples of non-polargroups are saturated or unsaturated hydrocarbons such as alkyl, alkenylor other lipid groups. Sterols (e.g., cholesterol) and otherpharmaceutically acceptable adjuvants (including anti-oxidants likealpha-tocopherol) may also be included to improve vesicle stability orconfer other desirable characteristics.

“Liposomes” are a subset of bilayer vesicles and are comprisedprincipally of phospholipid molecules which contain two hydrophobictails consisting of long fatty acid chains. Upon exposure to water,these molecules spontaneously align to form a bilayer membrane with thelipophilic ends of the molecules in each layer associated in the centerof the membrane and the opposing polar ends forming the respective innerand outer surface of the bilayer membrane. Thus, each side of themembrane presents a hydrophilic surface while the interior of themembrane comprises a lipophilic medium. These membranes when formed aregenerally arranged in a system of concentric closed membranes separatedby interlamellar aqueous phases, in a manner not dissimilar to thelayers of an onion, around an internal aqueous space. Thesemultilamellar vesicles (MLV) can be converted into small or unilamellarvesicles (UV), with the application of a shearing force.

“Cationic Liposome” is a Liposome that contains lipid components thathave an overall positive charge at physiological pH.

“SELEX” methodology involves the combination of selection of NucleicAcid Ligands which interact with a Target in a desirable manner, forexample binding to a protein, with amplification of those selectedNucleic Acids. Iterative cycling of the selection/amplification stepsallows selection of one or a small number of Nucleic Acids whichinteract most strongly with the Target from a pool which contains a verylarge number of Nucleic Acids. Cycling of the selection/amplifcationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology can be employed to obtain a NucleicAcid Ligand to a desirable Target.

The SELEX methodology is described in the SELEX Patent Applications.

“SELEX Target” means any compound or molecule of interest for which aligand is desired. A Target can be a protein (such as VEGF, thrombin,and selectin), peptide, carbohydrate, polysaccharide, glycoprotein,hormone, receptor, antigen, antibody, virus, substrate, metabolite,transition state analog, cofactor, inhibitor, drug, dye, nutrient,growth factor, etc. without limitation. The terms “SELEX Target” and“Target” can be used interchangeably herein. It will be clear from thesentence context whether or not “Target” means “SELEX Target.”

“Target” means a preselected location in a biological system includingtissues, organs, cells, intracellular compartments, extracellularcomponents. The latter include hormones (endocrine paracrine,autocrine), enzymes, neurotransmitters and constituents of physiologicalcascade phenomena (e.g., blood coagulation, complement, etc.).

“Improved Pharmacokinetic Properties” means that the Nucleic Acid Ligandin association with the Non-Immunogenic, High Molecular Weight Compoundor Lipophilic Compound shows a longer circulation half-life in vivorelative to the same Nucleic Acid Ligand not in association with aLipophilic Compound or Non-Immunogenic, High Molecular Weight Compoundor other pharmacokinetic benefits such as improved Target to non-Targetconcentration ratio.

“Linker” is a molecular entity that connects two or more molecularentities through covalent or Non-Covalent Interactions.

“Spacer” is a Linker of the size that allows spatial separation of twoor more molecular entities in a manner that preserves the functionalproperties of one or more of the molecular entities.

It is an object of the present invention to provide Complexes comprisingone or more Nucleic Acid Ligands in association with a LipophilicCompound or Non-Immunogenic, High Molecular Weight Compound. SuchComplexes have one or more of the following advantages over a NucleicAcid Ligand not in association with a Lipophilic Compound orNon-Immunogenic, High Molecular Weight Compound: 1) ImprovedPharmacokinetic Properties, 2) improved capacity for intracellulardelivery, or 3) improved capacity for targeting.

The Complexes of the present invention may benefit from one, two, or allthree of these advantages. The Complexes of the present invention maycontain different Nucleic Acid Ligands serving totally differentpurposes in the Complex. For example, a Complex of the present inventionmay be comprised of a) a Liposome, b) a Nucleic Acid Ligand that istargeted to an intracellular SELEX Target that is encapsulated withinthe interior of the Liposome, and c) a Nucleic Acid Ligand that istargeted to a particular cell type that is associated with andprojecting from the exterior surface of the Liposome. In such a case,the Complex will 1) have Improved Pharmacokinetic Properties due to thepresence of the Liposome, 2) have enhanced capacity for intracellulardelivery of the encapsulated Nucleic Acid Ligand due to the propertiesof the Liposome, and 3) be specifically targeted to the preselectedlocation ini vivo by the exteriorly associated Nucleic Acid Ligand.

In another embodiment, the Complex of the present invention is comprisedof a Nucleic Acid Ligand covalently attached to a Lipophilic Compoundsuch as cholesterol, dialkyl glycerol, diacyl glycerol, or aNon-Immunogenic, High Molecular Weight Compound such as polyethyleneglycol (PEG). In these cases, the pharmacokinetic properties of theComplex will be enhanced relative to the Nucleic Acid Ligand alone. Instill other embodiments, the Complex of the present invention iscomprised of a Nucleic Acid Ligand encapsulated inside a Liposome, andenhanced intracellular uptake of the Nucleic Acid Ligand is seen overthe un-Complexed Nucleic Acid Ligand.

In certain embodiments of the invention, the Complex of the presentinvention is comprised of a Nucleic Acid Ligand attached to one(dimeric) or more (multimeric) other Nucleic Acid Ligands. The NucleicAcid Ligand can be to the same or different SELEX Target. In embodimentswhere there are multiple Nucleic Acid Ligands to the same SELEX Target,there is an increase in avidity due to multiple binding interactionswith the SELEX Target. Furthermore, in embodiments of the inventionwhere the Complex is comprised of a Nucleic Acid Ligand attached to oneor more other Nucleic Acid Ligands, the pharmacokinetic properties ofthe Complex will be improved relative to one Nucleic Acid Ligand alone.

The Lipophilic Compound or Non-Immunogenic, High Molecular WeightCompound can be covalently bonded or associated through Non-CovalentInteractions with the Nucleic Acid Ligand(s). In embodiments where theLipophilic Compound is cholesterol, dialkyl glvcerol, diacyl glycerol,or the Non-Immunogenic, High Molecular Weight Compound is PEG, acovalent association with the Nucleic Acid Ligand(s) is preferred. Inembodiments where the Lipophilic Compound is a Cationic Liposome orwhere the Nucleic Acid Ligands are encapsulated within the Liposome, anon-covalent association with the Nucleic Acid Ligand(s) is preferred.In embodiments where covalent attachment is employed, the LipophilicCompound or Non-Immunogenic, High Molecular Weight Compound may becovalently bound to a variety of positions on the Nucleic Acid Ligand,such as to an exocyclic amino group on the base, the 5-position of apyrimidine nucleotide, the 8-position of a purine nucleotide, thehydroxyl group of the phosphate, or a hydroxyl group or other group atthe 5′ or 3′ terminus of the Nucleic Acid Ligand. Preferably, however,it is bonded to the 5′ or 3′ hydroxyl group thereof. Attachment of theNucleic Acid Ligand to other components of the Complex can be donedirectly or with the utilization of Linkers or Spacers.

The Lipophilic Compound or Non-Immunogenic, High Molecular WeightCompound can associate through Non-Covalent Interactions with theNucleic Acid Ligand(s). For example, in one embodiment of the presentinvention, the Nucleic Acid Ligand is encapsulated within the internalcompartment of the Lipophilic Compound. In another embodiment of thepresent invention, the Nucleic Acid Ligand associates with theLipophilic Compound through electrostatic interactions. For instance, aCationic Liposome can associate with an anionic Nucleic Acid Ligand.Another example of a Non-Covalent Interaction through ionic attractiveforces is one in which a portion of the Nucleic Acid Ligand hybridizesthrough Watson-Crick base-pairing or triple helix base pairing to anoligonucleotide which is associated with a Lipophilic Compound orNon-Immunogenic, High Molecular Weight Compound.

One problem encountered in the therapeutic and in vivo diagnostic use ofNucleic Acids is that oligonucleotides in their phosphodiester form maybe quickly degraded in body fluids by intracellular and extracellularenzymes such as endonucleases and exonucleases before the desired effectis manifest. Certain chemical modifications of the Nucleic Acid Ligandcan be made to increase the in vivo stability of the Nucleic Acid Ligandor to enhance or to mediate the delivery of the Nucleic Acid Ligand.Modifications of the Nucleic Acid Ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the Nucleic Acid Ligand bases or to the Nucleic AcidLigand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

Where the Nucleic Acid Ligands are derived by the SELEX method, themodifications can be pre- or post-SELEX modifications. Pre-SELEXmodifications yield Nucleic Acid Ligands with both specificity for theirSELEX Target and improved in vivo stability. Post-SELEX modificationsmade to 2′-OH Nucleic Acid Ligands can result in improved in vivostability without adversely affecting the binding capacity of theNucleic Acid Ligands. The preferred modifications of the Nucleic AcidLigands of the subject invention are 5′ and 3′ phosphorothioate cappingor 3′3′ inverted phosphodiester linkage at the 3′ end. For RNA ligands,additional 2′ amino (2′-NH₂) modification of some or all of thenucleotides is preferred.

In another aspect of the present invention, the association of theNucleic Acid Ligand with a Lipophilic Compound or Non-Immunogenic, HighMolecular Weight Compound, results in Improved PharmacokineticProperties (i.e., slower clearance rate) relative to the Nucleic AcidLigand not in association with a Lipophilic Compound or Non-Immunogenic,High Molecular Weight Compound. In one embodiment of the invention, theComplex includes a Lipid Construct. The Complex with the Nucleic AcidLigand can be formed through covalent or Non-Covalent Interactions. In apreferred embodiment, the Lipid Construct is a Lipid Bilayer Vesicle. Inthe most preferred embodiment, the Lipid Construct is a Liposome.

In certain embodiments of this invention, the Complex comprises aLiposome with a targeting Nucleic Acid Ligand projecting out of theLiposome. In embodiments where there are multiple Nucleic Acid Ligandsto the same Target, there is an increase in avidity due to multiplebinding interactions with the Target.

Liposomes for use in the present invention can be prepared by any of thevarious techniques presently known in the art or subsequently developed.Typically, they are prepared from a phospholipid, for example,distearoyl phosphatidylcholine, and may include other materials such asneutral lipids, for example, cholesterol, and also surface modifierssuch as positively charged (e.g., sterylamine or aminomannose oraminomannitol derivatives of cholesterol) or negatively charged (e.g.,dicetyl phosphate, phosphatidyl glycerol) compounds. MultilamellarLiposomes can be formed by the conventional technique, that is, bydepositing a selected lipid on the inside wall of a suitable containeror vessel by dissolving the lipid in an appropriate solvent, and thenevaporating the solvent to leave a thin film on the inside of the vesselor by spray drying. An aqueous phase is then added to the vessel with aswirling or vortexing motion which results in the formation of MLVs. UVscan then be formed by homogenization, sonication or extrusion (throughfilters) of MLV's. In addition, UVs can be formed by detergent removaltechniques.

In certain embodiments of this invention, the Complex comprises aLiposome with a targeting Nucleic Acid Ligand(s) associated with thesurface of the Liposome and an encapsulated therapeutic or diagnosticagent. Preformed Liposomes can be modified to associate with the NucleicAcid Ligands. For example, a Cationic Liposome associates throughelectrostatic interactions with the Nucleic Acid Ligand. Alternatively,a Nucleic Acid Ligand attached to a Lipophilic Compound, such ascholesterol, can be added to preformed Liposomes whereby the cholesterolbecomes associated with the liposomal membrane. Alternatively, theNucleic Acid Ligand can be associated with the Liposome during theformulation of the Liposome. Preferably, the Nucleic Acid Ligand isassociated with the Liposome by loading into preformed Liposomes.

It is well known in the art that Liposomes are advantageous forencapsulating or incorporating a wide variety of therapeutic anddiagnostic agents. Any variety of compounds can be enclosed in theinternal aqueous compartment of the Liposomes. Illustrative therapeuticagents include antibiotics, antiviral nucleosides, antifungalnucleosides, metabolic regulators, immune modulators, chemotherapeuticdrugs, toxin antidotes, DNA, RNA, antisense oligonucleotides, etc. Bythe same token, the Lipid Bilayer Vesicles may be loaded with adiagnostic radionuclide (e.g., Indium 111, Iodine 131, Yttrium 90,Phosphorous 32, or gadolinium) and fluorescent materials or othermaterials that are detectable in in vitro and in vivo applications. Itis to be understood that the therapeutic or diagnostic agent can beencapsulated by the Liposome walls in the aqueous interior.Alternatively, the carried agent can be a part of, that is, dispersed ordissolved in the vesicle wall-forming materials.

During Liposome formation, water soluble carrier agents may beencapsulated in the aqueous interior by including them in the hydratingsolution, and lipophilic molecules incorporated into the lipid bilayerby inclusion in the lipid formulation. In the case of certain molecules(e.g., cationic or anionic lipophilic drugs), loading of the drug intopreformed Liposomes may be accomplished, for example, by the methodsdescribed in U.S. Pat. No. 4,946,683, the disclosure of which isincorporated herein by reference. Following drug encapsulation, theLiposomes are processed to remove unencapsulated drug through processessuch as gel chromatography or ultrafiltration. The Liposomes are thentypically sterile filtered to remove any microorganisms which may bepresent in the suspension. Microorganisms may also be removed throughaseptic processing.

If one wishes to encapsulate large hydrophilic molecules with Liposomes,larger unilamellar vesicles can be formed by methods such as thereverse-phase evaporation (REV) or solvent infusion methods. Otherstandard methods for the formation of Liposomes are known in the art,for example, methods for the commercial production of Liposomes includethe homogenization procedure described in U.S. Pat. No. 4,753,788 andthe thin-film evaporation method described in U.S. Pat. No. 4,935,171,which are incorporated herein by reference.

It is to be understood that the therapeutic or diagnostic agent can alsobe associated with the surface of the Lipid Bilayer Vesicle. Forexample, a drug can be attached to a phospholipid or glyceride (aprodrug). The phospholipid or glyceride portion of the prodrug can beincorporated into the lipid bilayer of the Liposome by inclusion in thelipid formulation or loading into preformed Liposomes (see U.S. Pat.Nos. 5,194,654 and 5,223,263, which are incorporated by referenceherein).

It is readily apparent to one skilled in the art that the particularLiposome preparation method will depend on the intended use and the typeof lipids used to form the bilayer membrane.

A Nucleic Acid Ligand or ligands in association with a LipophilicCompound or Non-Immunogenic, High Molecular Weight Compound may enhancethe intracellular delivery of the Nucleic Acid Ligand(s) overnon-associated Nucleic Acid Ligand(s). The efficiency of delivery of theComplex to cells may be optimized by using lipid formulations andconditions known to enhance fusion of Liposomes with cellular membranes.For example, certain negatively charged lipids such asphosphatidylglycerol and phosphatidylserine promote fusion, especiallyin the presence of other fusogens (e.g., multivalent cations like Ca2+,free fatty acids, viral fusion proteins, short chain PEG, lysolecithin,detergents and surfactants). Phosphatidylethanolamine may also beincluded in the Liposome formulation to increase membrane fusion and,concomitantly, enhance cellular delivery. In addition, free fatty acidsand derivatives thereof, containing, for example, carboxylate moieties,may be used to prepare pH-sensitive Liposomes which are negativelycharged at higher pH and neutral or protonated at lower pH. SuchpH-sensitive Liposomes are known to possess a greater tendency to fuse.

In the preferred embodiment, the Nucleic Acid Ligands of the presentinvention are derived from the SELEX methodology. SELEX is described inU.S. patent application Ser. No. 07/536,428, entitled SystematicEvolution of Ligands by EXponential Enrichment, now abandoned, U.S.Patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitledNucleic Acid Ligands, now U.S. Pat. No. 5,475,096, U.S. patentapplication Ser. No. 07/931,473, filed Aug. 17, 1992, entitled NucleicAcid Ligands, now U.S. Pat. No. 5,270,163 (see also PCT/US91/04078).These applications, each specifically incorporated herein by reference,are collectively called the SELEX Patent Applications.

The SELEX process provides a class of products which are Nucleic Acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired Target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to Target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

-   -   1) A Candidate Mixture of Nucleic Acids of differing sequence is        prepared. The Candidate Mixture generally includes regions of        fixed sequences (i.e., each of the members of the Candidate        Mixture contains the same sequences in the same location) and        regions of randomized sequences. The fixed sequence regions are        selected either: (a) to assist in the amplification steps        described below, (b) to mimic a sequence known to bind to the        Target, or (c) to enhance the concentration of a given        structural arrangement of the Nucleic Acids in the Candidate        Mixture. The randomized sequences can be totally randomized        (i.e., the probability of finding a base at any position being        one in four) or only partially randomized (e.g., the probability        of finding a base at any location can be selected at any level        between 0 and 100 percent).    -   2) The Candidate Mixture is contacted with the selected Target        under conditions favorable for binding between the Target and        members of the Candidate Mixture. Under these circumstances, the        interaction between the Target and the Nucleic Acids of the        Candidate Mixture can be considered as forming Nucleic        Acid-target pairs between the Target and those Nucleic Acids        having the strongest affinity for the Target.    -   3) The Nucleic Acids with the highest affinity for the target        are partitioned from those Nucleic Acids with lesser affinity to        the target. Because only an extremely small number of sequences        (and possibly only one molecule of Nucleic Acid) corresponding        to the highest affinity Nucleic Acids exist in the Candidate        Mixture, it is generally desirable to set the partitioning        criteria so that a significant amount of the Nucleic Acids in        the Candidate Mixture (approximately 5-50%) are retained during        partitioning.    -   4) Those Nucleic Acids selected during partitioning as having        the relatively higher affinity for the target are then amplified        to create a new Candidate Mixture that is enriched in Nucleic        Acids having a relatively higher affinity for the target.    -   5) By repeating the partitioning and amplifying steps above, the        newly formed Candidate Mixture contains fewer and fewer unique        sequences, and the average degree of affinity of the Nucleic        Acids to the target will generally increase. Taken to its        extreme, the SELEX process will yield a Candidate Mixture        containing one or a small number of unique Nucleic Acids        representing those Nucleic Acids from the original Candidate        Mixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, entitled “Method for Selecting Nucleic Acids on theBasis of Structure”, describes the use of SELEX in conjunction with gelelectrophoresis to select Nucleic Acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands” describes a SELEX based method for selectingNucleic Acid Ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,entitled “High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine”, describes a method for identifying highlyspecific Nucleic Acid Ligands able to discriminate between closelyrelated molecules, termed Counter-SELEX. U.S. patent application Ser.No. 08/143,564, filed Oct. 25, 1993, entitled “Systematic Evolution ofLigands by EXponential Enrichment: Solution SELEX”, describes aSELEX-based method which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992,entitled “Methods of Producing Nucleic Acid Ligands” describes methodsfor obtaining improved Nucleic Acid Ligands after SELEX has beenperformed.

U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995,entitled “Systematic Evolution of Ligands by EXponential Enrichment:Chemi-SELEX”, describes methods for covalently linking a ligand to itstarget.

The SELEX method encompasses the identification of high-affinity NucleicAcid Ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified Nucleic Acid Ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides”, that describesoligonucleotides containing nucleotide derivatives chemically modifiedat the 5- and 2′-positions of pyrimidines. U.S. patent application Ser.No. 08/134,028, supra, describes highly specific Nucleic Acid Ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of 2′ Modified Pyrimidine Intramolecular NucleophilicDisplacement”, describes oligonucleotides containing various 2′-modifiedpyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. patent application Ser. No. 08/284,063, filed Aug.2, 1994. entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX” and U.S. patent application Ser. No.08/234,997, filed Apr. 28, 1994, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Blended SELEX”, respectively. Theseapplications allow the combination of the broad array of shapes andother properties, and the efficient amplification and replicationproperties, of oligonucleotides with the desirable properties of othermolecules. Each of the above described patent applications whichdescribe modifications of the basic SELEX procedure are specificallyincorporated by reference herein in their entirety.

SELEX identifies Nucleic Acid Ligands that are able to bind targets withhigh affinity and with outstanding specificity, which represents asingular achievement that is unprecedented in the field of Nucleic Acidsresearch. These characteristics are, of course, the desired propertiesone skilled in the art would seek in a therapeutic or diagnostic ligand.

In order to produce Nucleic Acid Ligands desirable for use as apharmaceutical, it is preferred that the Nucleic Acid Ligand (1) bindsto the target in a manner capable of achieving the desired effect on thetarget; (2) be as small as possible to obtain the desired effect; (3) beas stable as possible; and (4) be a specific ligand to the chosentarget. In most situations, it is preferred that the Nucleic Acid Ligandhas the highest possible affinity to the target. Additionally, NucleicAcid Ligands can have facilitating properties.

In co-pending and commonly assigned U.S. patent application Ser. No.07/964,624, filed Oct. 21, 1992 ('624), methods are described forobtaining improved Nucleic Acid Ligands after SELEX has been performed.The '624 application, entitled Methods of Producing Nucleic AcidLigands, is specifically incorporated herein by reference.

In embodiments where the Nucleic Acid Ligand(s) can serve in a targetingcapacity, the Nucleic Acid Ligands adopt a three dimensional structurethat must be retained in order for the Nucleic Acid Ligand to be able tobind its target. In addition, the Nucleic Acid Ligand must be properlyoriented with respect to the surface of the Complex so that its Targetbinding capacity is not compromised. This can be accomplished byattaching the Nucleic Acid Ligand at a position that is distant from thebinding portion of the Nucleic Acid Ligand. The three dimensionalstructure and proper orientation can also be preserved by use of aLinker or Spacer as described supra.

Any variety of therapeutic or diagnostic agents can be attached,encapsulated, or incorporated into the Complex as discussed supra fortargeted delivery by the Complex. In embodiments where the Complex iscomprised of a Liposome and a Nucleic Acid Ligand, for example, afungi-specific Nucleic Acid Ligand exposed on the surface of the Complexcould Target a fungal cell for delivery of a fungicide (e.g.,amphotericin B). Alternatively, a chemotherapeutic agent can bedelivered to tumor cells via a Nucleic Acid Ligand to a tumor antigen.

In an alternative embodiment, the therapeutic or diagnostic agent to bedelivered to the Target cell could be another Nucleic Acid Ligand. Forexample, a Nucleic Acid Ligand that binds to a tumor antigen could bepresented to the outside of the Complex, and a Nucleic Acid Ligand thatbinds to and inhibits the mutated isoform of an intracellular Targetsuch as p21, the protein product of the ras gene, could be the agent tobe delivered.

It is further contemplated by this invention that the agent to bedelivered can be incorporated into the Complex in such a way as to beassociated with the outside surface of the Complex. (e.g., a prodrug,receptor antagonist, or radioactive substance for treatment or imaging).As with the Nucleic Acid Ligand, the agent can be associated throughcovalent or Non-Covalent Interactions. The Complex would providetargeted delivery of the agent extracellularly, with the Liposomeserving as a Linker.

In another embodiment, a Non-Immunogenic, High Molecular Weight Compound(e.g., PEG) can be attached to the Liposome to provide ImprovedPharmacokinetic Properties for the Complex. Nucleic Acid Ligands may beattached to the Liposome membrane as described supra or may be attachedto a Non-Immunogenic, High Molecular Weight Compound which in turn isattached to the membrane. In this way, the Complex may be shielded fromblood proteins and thus be made to circulate for extended periods oftime while the Nucleic Acid Ligand is still sufficiently exposed to makecontact with and bind to its SELEX Target.

In one embodiment of this invention, the Nucleic Acid Ligand presentedon the outside of the Complex can Target circulating proteins (e.g.,antibodies, growth factors, protein hormones) for removal by thereticuloendothelial system (i.e., liver and spleen). As an example, thetreatment of autoimmune diseases may be possible by such a Complex.Autoimmune diseases are the result of a failure of an organism's immunesystem to avoid recognition of self due to production of autoantibodiesand autoreactive T cells. The attack by the immune system on host cellscan result in a large number of disorders including neural diseases,such as multiple sclerosis and myasthenia gravis; diseases of thejoints, such as rheumatoid arthritis; attacks on Nucleic Acids, asobserved with systemic lupus erythematosus; and such other diseasesassociated with various organs, as psoriasis, juvenile onset diabetes,Sjogren's disease, and Graves disease. As it has been found thatLiposomes associated with proteins are generally cleared by thereticuloendothelial system (i.e., spleen and liver) faster thanLiposomes without associated proteins, Nucleic Acid Ligands complexedwith a Liposome, can be used for removal of autoantibodies by thereticuloendothelial system.

In another embodiment of the present invention, Nucleic Acid Ligandsspecific for the same SELEX Target are attached to the surface of thesame Liposome. This provides the possibility of bringing the same SELEXTargets in close proximity to each other and can be used to generatespecific interactions between the same SELEX Targets. For example,Nucleic Acid Ligands to a tyrosine kinase receptor attached to aLiposome would bring the receptors in close proximity to one another.This would facilitate autophosphorylation which would initiate a signaltransduction cascade.

In an alternative embodiment of the present invention, Nucleic AcidLigands specific for different SELEX Targets are attached to the surfaceof the same Liposome. This provides the possibility of bringing thedistinct Targets in close proximity to each other and can be used togenerate specific interactions between the Targets. For example, NucleicAcid Ligands specific for a tumor marker or antigen and Nucleic AcidLigands specific for a T-cell receptor would bring the T-cells in closeproximity to the tumor. In addition to using the Liposome as a way ofbringing Targets in close proximity, agents could be encapsulated in theLiposome (e.g., immune system modulator) to increase the intensity ofthe interaction (e.g., increase the T-cell immune response).

In instances where it is difficult to identify biomolecules that areunique to a cellular Target of interest, specificity may be obtained byhaving Nucleic Acid Ligands that are specific for two or more markers tothe Target associated with the Complex. In this scenario, it is expectedthat the best Nucleic Acid Ligands would have low or medium affinity fortheir respective Targets. The use of Nucleic Acid Ligands of this typeare recommended since high affinity Nucleic Acid Ligands would lead tothe association of drug with all cells possessing either marker protein,thereby reducing specificity. With lower affinity ligands, avidity isrequired to provide the necessary specificity.

The Liposome/Nucleic Acid Ligand Complex also allows for the possibilityof multiple binding interactions to the Target. This, of course, dependson the number of Nucleic Acid Ligands per Complex and mobility of theNucleic Acid Ligands and receptors in their respective membranes. Sincethe effective binding constant may increase as the product of thebinding constant for each site, there is a substantial advantage tohaving multiple binding interactions. In other words, by having manyNucleic Acid Ligands attached to the Liposome, and therefore creatingmultivalency, the effective affinity (i.e., the avidity) of themultimeric Complex for its Target may become as good as the product ofthe binding constant for each site.

In certain embodiments of the invention, the Complex of the presentinvention is comprised of a Nucleic Acid Ligand attached to a LipophilicCompound such as cholesterol, or dialkyl glycerol, or diacyl glycerol.In this case, the pharmacokinetic properties of the Complex will beimproved relative to the Nucleic Acid Ligand alone. As discussed supra,cholesterol may be covalently bound to the Nucleic Acid Ligand atnumerous positions on the Nucleic Acid Ligand. In another embodiment ofthe invention, the Complex may further comprise a Lipid Construct suchas a Liposome. In this embodiment, the cholesterol can assist in theincorporation of the Nucleic Acid Ligand into the Liposome due to thepropensity for cholesterol to associate with other Lipophilic Compounds.The cholesterol in association with a Nucleic Acid Ligand can beincorporated into the lipid bilayer of the Liposome by inclusion in theformulation or by loading into preformed Liposomes. In the preferredembodiment, the cholesterol/Nucleic Acid Ligand Complex is associatedwith a preformed Liposome. The cholesterol can associate with themembrane of the Liposome in such a way so as the Nucleic Acid Ligand isprojecting into or out of the Liposome. In embodiments where the NucleicAcid Ligand is projecting out of the Complex, the Nucleic Acid Ligandcan serve in a targeting capacity.

In other embodiments, the Complex of the present invention is comprisedof a Nucleic Acid Ligand attached to a Non-Immunogenic, High MolecularWeight Compound such as PEG, dialkyl glycerol, diacyl glycerol, orcholesterol. In this embodiment, the pharmacokinetic properties of theComplex are improved relative to the Nucleic Acid Ligand alone. Asdiscussed supra, the association could be through Covalent Bonds orNon-Covalent Interactions. In the preferred embodiment, the Nucleic AcidLigand is associated with the PEG, dialkyl glycerol, diacyl glycerol, ora cholesterol molecule through Covalent Bonds. Also, as discussed supra,where covalent attachment is employed, PEG, dialkyl glycerol, diacylglycerol, or cholesterol may be covalently bound to a variety ofpositions on the Nucleic Acid Ligand. In embodiments where PEG or diacylglycerol are used, it is preferred that the Nucleic Acid Ligand isbonded to the 5′ thiol through a maleimide or vinyl sulfonefunctionality or via a phosphodiester linkage. In embodiments wheredialkyl glycerol and cholesterol are used, it is preferred that theNucleic Acid Ligand is bonded via a phosphodiester linkage. In certainembodiments, a plurality of Nucleic Acid Ligands can be associated witha single PEG, dialkyl glycerol, diacyl glycerol, or cholesterolmolecule. The Nucleic Acid Ligands can be to the same or differentTarget. In embodiments where there are multiple Nucleic Acid Ligands tothe same Target, there is an increase in avidity due to multiple bindinginteractions with the Target. In yet further embodiments, a plurality ofPEG, dialkyl glycerol, diacyl glycerol, or cholesterol molecules can beattached to each other. In these embodiments, one or more Nucleic AcidLigands to the same Target or different Targets can be associated witheach PEG, dialkyl glycerol, diacyl glycerol, or cholesterol molecule.This also results in an increase in avidity of each Nucleic Acid Ligandto its SELEX Target. In embodiments where multiple Nucleic Acidsspecific for the same SELEX Target are attached to PEG, dialkylglycerol, diacyl glycerol, or cholesterol, there is the possibility ofbringing the same Targets in close proximity to each other in order togenerate specific interactions between the same Targets. Where multipleNucleic Acid Ligands specific for different Targets are attached to PEG,dialkyl glycerol, diacyl glycerol, or cholesterol, there is thepossibility of bringing the distinct Targets in close proximity to eachother in order to generate specific interactions between the Targets. Inaddition, in embodiments where there are Nucleic Acid Ligands to thesame Target or different Targets associated with PEG, dialkyl glycerol,diacyl glycerol, or cholesterol, a drug can also be associated with PEG,dialkyl glycerol, diacyl glycerol, or cholesterol. Thus the Complexwould provide targeted delivery of the drug, with PEG, dialkyl glycerol,diacyl glycerol, or cholesterol serving as a Linker.

In another embodiment of the invention, the Complex is comprised of aNucleic Acid Ligand attached to a Non-Immunogenic, High Molecular WeightCompound such as magnetite. As discussed supra, the association could bethrough Covalent Bonds or Non-Covalent Interactions. In the preferredembodiment, the Nucleic Acid Ligand is associated with magnetite throughCovalent Bonds. The magnetite can be coated with a variety of compoundsthat display different functional chemistries for attachment (e.g.,dextran, Lipophilic Compounds). The Nucleic Acid Ligand in associationwith the magnetite provides targeted delivery of the magnetite for usein nuclear magnetic resonance imaging.

The following examples are provided to explain and illustrate thepresent invention and are not to be taken as limiting of the invention.The structures of the Nucleic Acid Ligands described in the examplesbelow are shown in FIG. 1. Example 1 describes the conjugation ofNucleic Acid Ligands with lipid, dialkyl glycerol or diacyl glycerol, aswell as incorporation of pharmacokinetic modifiers via automatedsynthesis. Example 2 describes the conjugation of PEG and cholesterolwith a Nucleic Acid Ligand. The modifications to the Nucleic Acid Liganddo not interfere with its ability to bind to its SELEX Target, as thebinding affinities of the PEG-conjugated and cholesterylated NucleicAcid Ligands were identical to the non-conjugated andnon-cholesterylated molecules. Example 3 describes the incorporation ofa cholesterol-derivatized Nucleic Acid Ligand into a lipid formulation.The activity of the Nucleic Acid Ligand/Liposome formulations containingthrombin Nucleic Acid Ligands was tested in an in vitro clottinginhibition assay. Liposome processing conditions do not affect theanticoagulation activity of the Nucleic Acid Ligand. In addition, theliposomal association does not affect the ability of the Nucleic AcidLigand to bind and inhibit its Target. Example 4 describes thepharmacokinetic properties of Nucleic Acid Ligands in association withcholesterol alone, dialkyl glycerol alone, with PEG alone, withcholesterol and Liposome, with dialkyl glycerol and Liposomes, and withPEG and Liposome. Nucleic Acid Ligands that have been modified at the 2′sugar position of purines and pyrimidines are also included. Example 5reports on the toxicity and intracellular uptake by human lymphocytes ofCationic Liposome-Nucleic Acid Ligand Complexes. Examples 6-10 describethe following effects on the incorporation of Nucleic Acid Ligands intopreformed Liposomes: varying the negative charge of the lipids, varyingthe cholesterol content, varying the lipid/Nucleic Acid Ligand ratiowith a fixed amount of Nucleic Acid Ligand, varying the lipid/NucleicAcid Ligand ratio with a fixed amount of SUV, and varying thephospholipid chain length. Example 11 demonstrates that incorporation ofa Nucleic Acid Ligand/cholesterol conjugate into a liposomal formulationhas occurred via non-denaturing gel electrophoresis. Example 12describes the way in which Nucleic Acid Ligands can be passivelyencapsulated into Liposomes. Example 13 describes the way in whichNucleic Acid Ligands can be remotely loaded into Liposomes. Example 14describes the covalent conjugation of Nucleic Acid Ligands to Liposomes.Example 15 describes the in vitro and in vivo efficacy of a Nucleic AcidLigand-Liposome Complex.

EXAMPLE 1

Lipid, Peg, Dialkyl Glycerol and Diacyl Glycerol Reagents forOligonucleotide Modification

In this example, conjugation of Nucleic Acid Ligands with lipid and/orPEG or diacyl glycerol or diakyl glycerol reagents is described, as wellas incorporation of the pharmacokinetic modifiers via automatedsynthesis using either phosphoramidite or H-phosphonate couplingchemistry. In the schemes depicted below, a solid arrow represents stepsthat have been completed, whereas a dashed arrow represents steps thathave not yet been completed. Scheme 1 depicts the preparation of adipalmitoyl phosphatidyl ethanolamine maleimide reagent for coupling tosulfhydryl-modified oligonucleotide substrates. This procedure isanalogous to that reported by Cheronis et al (Cheronis, J. C. et al., J.Med. Chem. (1992) 35:1563-1572) for the preparation of bis- andtris-maleimide reagents from simple, aliphatic di- and triaminesubstrates. Treatment of the phospholipid with methoxycarbonyl maleimideresulted in formation of an uncharacterized intermediate which, uponincubation with sulfhydryl-modified oligonucleotide, resulted incomplete conversion of the oligonucleotide to the dipalmitoylphosphatidyl ethanolamine conjugate (vida infra). Similar reagents arealso available commercially from Avanti Polar Lipids.

The ability to modify oligonucleotides under automated synthesisconditions has many obvious advantages. Reagents were prepared whichallowed facile incorporation of lipid and/or PEG moieties under standardautomated synthesis conditions. Initially, the versatile module 10 wasdesigned and synthesized (Scheme 2) which could be divergently convertedto any number of oligo modification reagents of interest by simplychemoselectively functionalizing the amine group then phosphitylation ofthe hydroxyl group. Noteworthy is the flexibility this strategy offerswith respect to the modification group (the amine ligand) and theactivated phosphate precursor (phosphoramidite, H-phosphonate, orphosphate triester) introduced via derivatization of the 2′- hydroxylgroup. Additionally, the glycerol nucleus of automated synthesisreagents prepared in this way renders the products suitable for oligomodification at internal chain positions, or at the 5′ end. Analternative synthesis of module 10 is shown in Scheme 3. Tetraethyleneglycol (1; TEG) is derivatized as the monotosylate 2a upon treatmentwith a limiting amount of p-toluenesulfonyl chloride, preferably 10 molepercent, in a basic medium, preferably pyridine. In this manner, 2a wasobtained in 75% yield after silica gel filtration. Conversion of 2a tothe TEG phtalimide 3a was accomplished in 80% yield upon treatment withphthalimide in the presence of diazabicycloundecane (DBU) as a base atelevated temperature in DMF solution. Allylation of phthalimide 3a(allyl bromide, NaH, THF/DMF) afforded 65% yield of allyl TEG 4a.Treatment of 4a with 0.5% OsO₄ and 1.1 equivalents of N-methylmorpholineN-oxide (NMO) afforded a diol intermediate that was, without furtherpurification, converted to the dimethoxytrityl (DMT) ether derivative 9in 89% overall yield for the two steps. Finally, amine deprotectionusing 40% MeNH₂ was carried out to afford 10 in 95% purified yield.Module 10 has been further elaborated by treatment withPEG-nitrophenylcarbonate (PEG-NPC; Shearwater Polymers). In this way,the phosphitylation precursor 12 (Scheme 4) was prepared in excellentyield. Further conversions of 12 to both phosphoramidite 13 andH-phosphonate 14 have been carried out.

The design of a lipid reagent for oligonucleotide modification byautomated synthesis necessitates replacement of the ester linkages ofnative diacyl glycerols, as in the dipalmitoyl phosphatidyl derivativedescribed above, by glycerol-alkyl linkages stable to the basicdeprotection protocol required for synthetic oligo recovery. The linkagethat was chosen to explore initially was the ether linkage, as in theknown dipalmityl glycerol derivative 15 (available from Sigma), althoughlong-chain alkyl carbanates (or a combination of ethers and carbamates)would also be suitable. Dipalmityl glycerol was activated as the acylcarbonyl imidazole (CDI, pyridine) and this activated intermediate wascoupled with the module 10 (pyridine,. 80° C.; 44%). Phosphitylation(CIP(iPr₂N)OCH₂CH₂CN; diisopropylethylamine (DIPEA), CH₂Cl₂; 59%) of 16afforded the phosphoramidite 17 (Scheme 5). Synthesis of a C₁₈ analog ofamidite 17 via a chloroformate intermediate is shown in Scheme 6. Thedialkyl glycerol (18; DAG) was converted to the correspondingchloroformate 19 upon treatment with excess phosgene in toluene.Conjugation of 19 and amino alcohol 10 was carried out in pyridine toafford adduct 20 in 57% purified yield. Phosphitylation of the secondaryhydroxyl of 20 under standard condition afforded phosphoramidite 21 in95% yield. Coupling of amidite 17 to the 5′-end of a trinucleotide (TTT)on an ABI 394 automated oligonucleotide synthesizer using a slightlymodified synthesis cycle with extended coupling times (2×30 mincouplings) for the lipid amidite resulted in 94+% coupling efficiency,as determined by in-line trityl cation analysis.

Example A

Synthesis of Dipalmitoyl Phosphatidylethanolamine (DPPE)

Maleimide Reagent: A suspension of 200 mg (0.289 mmol) of DPPE and 54 mg(0.347 mmol) of methoxycarbonyl maleimide in 10 mL of THF/saturatedNaHCO₃ solution (1:1) was stirred at ambient temperature. After 12 h,the mixture was treated with 100 mL of EtOAc and the organic phase(which contained a gelatinous suspension of the product) separated fromthe aqueous phase. The organic phase was concentrated in vacuo,coevaporated twice with MeOH, and the resultant white solid trituratedthree times with EtOAc. This material was used without furthercharacterization or purification in oligonucleotide conjugationexperiments (vida infra).

Example B

Synthesis and Elaborations of Automated Synthesis Module 10.

Tetraethylene glycol dimethoxytrityl ether (2): Tetraethylene glycol(76.4 mL, 0.44 mol) was dissolved in 300 mL of anhydrous pyridine andcooled to 0° C. 4,4′-dimethoxytrityl chloride (15 g, 0.044 mol) wasadded as a solid with stirring. The reaction flask was covered with adrying tube and the reaction was allowed to warm to ambient temperatureovernight. The reaction was concentrated in vacuo at low temperature(<30° C.). The residue was diluted with 300 mL of ethyl acetate andextracted with 3×300 mL of water. The combined aqueous layers wereextracted with ethyl acetate, and the combined organic layers were driedover sodium sulfate and concentrated. The crude residue was purified byflash chromatography using 1000 mL of silica gel (wet-packed onto columnwith hexane containing 5% triethylamine), eluting with 10-20-40-60-80%ethyl acetate in hexane containing 5% triethylamine, and then ethylacetate containing 5% triethylamine. 19.51 g (89%) of 2 was collected asa gold oil. ¹H NMR (300 MHz, CDCl3) d 7.47-7.16 (overlapping signals,9H), 6.79 (d, 4H), 3.72 (s, 6H), 3.66-3.62 (m, 2H), 3.22 (t, J=5.22 Hz,1H), 2.96 (br t, 1H); ¹³C NMR (75 MHz, CDCl₃) d 158.12, 144.86, 136.04,129.81, 127.93, 127.49, 126.40, 112.78, 85.67, 72.31, 70.48, 70.44,70.12, 62.89, 61.39, 54.89; Low resolution MS m/e calculated forC₁₅H₂₅O₇S (M−DMT+1⁺): 349.167, found 349.1.

Tetraethylene glycol dimethoxytrityl ether p-toluenesulfonate (3):Compound 2 ( 5.0 g, 10.06 mmol) was dissolved in 50 mL of anhydrousdichloromethane and cooled to 0° C. Triethylamine (1.82 mL, 13.1 mmol)was added, followed by p-toluenesulfonyl chloride (1.92 g, 10.06 mmol)as a solid, with stirring. The reaction was stored in the refrigeratorovernight. TLC Analysis indicated the reaction was approximately 80%complete. An additional 0.5 equivalents of triethylamine and 0.5equivalents of p-toluenesulfonyl chloride were added, and the reactionwas stirred at room temperature overnight. The reaction was filteredthrough Celite and concentrated. The residue was purified by flashchromatography using 300 mL of silica gel (wet-packed onto column using5% triethylamine in hexane) eluting with 25-50-75% ethyl acetate inhexane containing 5% triethylamine, and then ethyl acetate containing 5%triethylamine. 5.7 g (87%) of 3 was collected as an orange oil. ¹H NMR(300 MHz, CDCl₃) d 7.75 (d, 2H), 7.44-7.12 (m, 11H), 6.78 (d, 4H),4.12-4.09 (m, 2H), 3.73 (s, 6H), 3.66-3.54 (m, 13H), 3.22 (t, J=3.87 Hz,2H), 2.41 (s, 3H).

Tetraethylene glycol monotosylate (2a): Tetraethylene glycol (200 mL,1.15 mol) was dissolved in 500 mL of pyridine and cooled to 0° C. andtreated with 22.0 g (0.115 mol) of p-toluenesulfonyl chloride. Whensolution was complete, the reaction mixture was stored in therefrigerator overnight, and then concentrated in vacuo. The residue wasdissolved in 800 mL of EtOAc and extracted with 3×600 mL of H₂O. The H₂Ofractions were back-extracted with EtOAc, and the combined EtOAcfractions were extracted with saturated aqueous Na₂HPO₄. The organicphase was dried over MgSO₄ and concentrated to a colorless oil The oilwas purified by flash chromatography using 800 mL of silica gel andeluting with hexane, 25% EtOAc-50% EtOAc in hexane, then EtOAc, then 10%MeOH-20% MeOH in EtOAc to afford 23.7 g (60%) of pure product and 11% ofproduct containing a minor impurity. 2a: ¹H NMR (300 MHz, CDCl₃) d 7.77(d, J=8.1 Hz, 2H), 7.32 (d, J=8.1 Hz, 2H), 4.13 (t, J=4.8 Hz, 2H),3.68-3.53 (m, 14H), 2.58 (t, J=5.6 Hz, 1H), 2.42 (s, 3H); ¹³C NMR (75MHz, CDCl₃) d 168.2, 158.3, 144.8, 135.9, 133.8, 132.0, 129.9, 128.0,127.7, 126.6, 123.1, 113.0, 85.9, 73.0, 70.6, 70.4, 70.0, 69.7,67.8,64.4, 55.1,37.1; Low resolution MS m/e calculated for C₁₅H₂₄0₈S(M+1): 349.1.

Tetraethylene glycol monophthalimide (3a): To a stirred solution of31.96 g (0.092 mol) of 2a in 400 mL of anhydrous DMF was added 14.2 g(1.05 equiv.) of phthalimide and 14.4 mL (1.05 equiv.) of1,8-diazabicyclo[5.4.0]undec-7-ene. The solution was heated at 70° C.for 18 h then concentrated in vacuo. The crude yellow oil was purifiedby flash chromatography using 1600 mL of silica gel and eluting with 25%EtOAc-50% EtOAc-75% EtOAc in hexane, then EtOAc, then 10% MeOH-20% MeOHin EtOAc to afford 23.8 g (80%) of 3a as an oil. Upon standing, 3abecame a waxy white solid. ¹H NMR (300 MHz, CDCl₃) d 7.84-7.78 (m, 2H),7.70-7.66 (m, 2H), 3.86 (t, J=5.6 Hz, 2H), 3.70 (t, J=5.6 Hz, 2H),3.64-3.51 (m, 12H), 2.67 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) d 168.2,133.8, 132.0, 123.1, 72.4, 70.5, 70.4, 70.2, 70.0, 67.8, 61.6, 37.2.

Synthesis of compound 4a: A solution of 15 g (0.0464 mol) of 3a in 150mL of THF and 15 mL of DMF was cooled to 0° C. under Ar. Allyl bromide(6.0 mL, 1.5 equiv.) was added to the solution, followed by addition of1.76 g (1.5 equiv.) of NaH as a solid. The opaque yellow suspension wasstirred at 0° C. for 30 minutes and then at room temperature for 18 hr.MeOH (50-100 mL) was added and concentrated then mixture wasconcentrated in vacuo. The crude material was purified by flashchromatography using 1500 mL of silica gel and eluting with 25%EtOAc-50% EtOAc-75% EtOAc in hexane, then EtOAc, then 10% MeOH in EtOActo afford 11.05 g (65%) of 4a as a yellow oil. ¹H NMR (300 MHz, CDCl₃) d7.84-7.80 (m, 2H), 7.72-7.67 (m, 2H), 5.94-5.84 (m, 1H), 5.28-5.14 (m,2H), 3.99 (d, J=5.61 Hz, 2H), 3.88 (t, J=5.85 Hz, 2H), 3.72 (t, J=5.76Hz, 2H), 3.64-3.54 (m, 13H); ¹³C NMR (75 MHz, CDCl₃) d 168.0, 134.6,133.7, 131.9, 123.0, 116.9, 72.0, 70.4, 69.9, 69.2, 67.7, 37.0.

(S)-(+)-2,2-Dim ethyl-1,3-dioxolanyl-4-ylmethyl(dimethoxytrityl)tetra-ethylene glycol (5): Sodium hydride (0.56 g, 23.5mmol) is weighed into a flame-dried flask, and 70 mL of anhydroustetrahydrofuran and 15 mL of anhydrous N,N-dimethylformamide were added.The suspension was cooled to 0° C. under argon, and(S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (2.7 mL, 21.7 mmol) wasadded dropwise via syringe. After stirring for 30 min at 0° C., compound3 (11.77 g, 18.1 mmol) in 15 mL of tetrahydrofuran was added dropwisevia addition funnel. The reaction mixture was stirred at ambienttemperature overnight, then quenched with 100 mL of saturated aqueoussodium bicarbonate and diluted with 300 mL of diethyl ether. The layerswere separated, and the ether layer was extracted 3 times with 300 mL ofwater. The ether layer was dried over sodium sulfate and concentrated.The residue was purified by flash chromatography using 500 mL of silicagel and eluting first with hexane and then 10-20-30-40-50-75% ethylacetate in hexane and then with ethyl acetate. 8.93 g (82%) of 5 wascollected as a colorless oil. ¹H NMR (CDCl₃) d 7.46-7.43 (m, 2H),7.34-7.17 (m, 7H), 6.78 (d, 4H), 4.23 (pentet, J=6.1 Hz, 1H), 4.00 (t,8.2H), 3.75 (s, 6H), 3.71-3.60 (m, 1SH), 3.53 (dd, J=10.0, 5.7 Hz, 1H),3.52 (10.4, J=5.2 Hz, 1H), 1.39 (s, 3H), 1.33 (s, 3H); ¹³C NMR (75 MHz,CDCl₃) d 158.24, 144.97, 136.20, 129.93, 128.07, 127.61, 126.51, 112.89,109.21, 85.77, 74.57, 72.20, 70.82, 70.59, 70.40, 69.68, 66.67, 63.01,55.04, 26.67, 25.29; Low resolution MS m/e calculated for C₃₅H₅₀O₉N(M+NH₄ ⁺): 628.399, found 628.5.

(S)-(+)-2,2-Dimethyl-1,3-dioxolanyl-4-ylmethyl tetraethylene glycol (6):100 mL of 80% acetic acid was cooled to 0° C. and then added to compound5 (6.6 g, 10.8 mmol) The clear orange solution was stirred at 0° C. for1 hr. Methanol (100 mL) was added, and the reaction mixture wasconcentrated in vacuo at low temperature (<30° C.). The residue waspurified by flash chromatography using 200 mL of silica gel, elutingfirst with ethyl acetate, and then 5-10-15-20% methanol in ethylacetate. 2.5 g (74%) of 6 was collected as a colorless oil. ¹H NMR(CDCl₃) d 4.21 (pentet, J=6.2 Hz, 1H), 4.08 (dd, J=5.9, 4.8 Hz, 1H),3.82-3.35 (m, 19H), 2.93 (br s, 1H), 1.34 (s, 3H), 1.28 (s, 3H); ¹³C NMR(75 MHz, CDCl₃) d 109.20, 74.50, 72.42, 72.14, 70.76, 70.39, 70.32,70.14, 66.63, 61.48, 26.61, 25.23; Low resolution MS m/e calculated forC₃₅H₅₀O₉N (M+NH₄ ⁺): 628.399, found 628.5.

(S)-(+)-2,2-Dimethyl-1,3-dioxolanyl-4-ylmethyl(phthalimido)tetraethylene glycol (8): Alcohol 6 ( 4.06 g, 13.2 mmol)was dissolved in 50 mL of anhydrous dichloromethane and cooled to 0° C.Triethylamine (3.7 mL, 26.3 mmol) was added, followed by addition ofp-toluenesulfonyl chloride (3.26 g, 17.1 mmol). The reaction flask wascovered by a drying tube and allowed to warm to room temperatureovernight. The reaction mixture was filtered through Celite, and thefiltrate was concentrated in vacuo. The crude material was purified byflash chromatography on 400 mL of silica gel, eluting first with 10%ethyl acetate in hexane, and then 20-40-60-80-100% ethyl acetate, andthen 10% methanol in ethyl acetate. Collected 5.21 g (85%) of theintermediate tosylate as a gold oil. (¹H NMR (400 MHz, CDCl₃) d 7.79 (d,J=8.1 Hz, 2H, tosyl aromatics), 7.32 (d, J=8.1 Hz, 2H, tosyl aromatics),4.25 (pentet, J=6.0 Hz, 1H), 4.13 (t, 4.7H), 4.02 (dd, J=8.12, 6.4 Hz,1H), 3.71-3.40 (m, 18H), 2.42 (s, 3H), 1.46 (s, 3H), 1.34 (s, 3H); ¹³CNMR (75 MHz, CDCl₃) d 144.74, 133.1, 129.76, 127.91, 109.8, 74.63,72.27, 70.89, 70.68, 70.52, 70.44, 69.19, 68.60, 66.74, 64.1, 26.73,25.34, 21.59; Low resolution MS m/e calculated for C₂₁H₃₈O₉NS (M+NH₄ ⁺):480.364, found 480.2.) The tosylate (5.2 g, 11.24 mmol) was dissolved in60 mL of anhydrous dimethylformamide. 1,8-Diazabicyclo-[5.4.0]undec-7-ene (1.7 mL, 11.24 mmol) was added, followed by phthalimide (1.65 g,11.24 mol). The reaction mixture was heated to 70° C. overnight. Thereaction was concentrated in vacuo, and purified by flash chromatographyon 400 mL of silica gel, eluting with 50% ethyl acetate in hexane.Collected 3.96 g (81%) of 8 as a colorless oil. ¹H NMR (300 MHz, CDCl₃)d 7.83-7.79 (m, 2H), 7.72-7.68 (m, 2H), 4.26 (pentet, J=6.0 Hz, 1H),4.03 (dd, J=8.2, 6.5 Hz, 1H), 3.88 (t, J=5.8 Hz, 1H), 3.74-3.44 (m,18H), 1.39 (s, 3H), 1.33 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) d 168.21,133.88, 132.10, 123.19, 109.33, 74.66, 72.30, 70.90, 70.52. 70.04,67.87, 66.79, 37.19, 26.75, 25.37; MS m/e calculated for C₂₂H₃₅O₈N₂(M+NH₄ ⁺): 455.288, found 455.2.

1-Dimethoxytrityl-3-(phthalimidotetraethylene glycolyl)-sn-glycerol (9):According to Scheme 2, compound 9 is synthesized as follows: the acetyl8 (5.16 g. 11.8 mmol) was dissolved in 100 mL of anhydrous methanol, andanhydrous p-toluenesulfonic acid (100 mg) was added. The reaction flaskwas covered with a drying tube and the reaction was stirred at ambienttemperature for 2.5 h, then neutralized by the addition of 10 mL ofanhydrous pyridine, concentrated in vacuo, and coevaporated withanhydrous pyridine. The resulting diol was then dissolved in 150 mL ofanhydrous pyridine and cooled to 0° C. 4,4′-Dimethoxytrityl chloride(4.39 g, 13 mmol) was added as a solid. The reaction flask was coveredwith a drying tube, and the reaction was allowed to warm to ambienttemperature overnight. Methanol (50 mL) was added, and the reaction wasconcentrated in vacuo. The crude material was purified by flashchromatography on 700 mL of silica gel (wet-packed onto column with 5%triethylamine in hexane), eluting first with 10% ethylacetate in hexane(containing 5% triethylamine) and then 20-40-60-80-100% ethyl acetate(containing 5% triethylamine). Collected 6.98 g (82%) of 9 as a paleyellow oil. ¹H NMR (300 MHz, CDCl₃) d 7.80 (dd, J=5.4, 3.1 Hz, 2H), 7.68(dd, J=5.4, 3.1 Hz, 2H), 7.42-7.14 (m, 9H, DMT), 6.79 (d, 4H, DMT), 3.95(br m, 1H), 3.86 (t, J=5.9 Hz, 1H), 3.75 (s, 6H), 3.70 (t, J=5.6 Hz,1H), 3.63-3.37 (m, 18H), 3.16 (m, 2H), 2.84 (br d, 1H); ¹³C NMR (75 MHz,CDCl₃) d 168.15, 158.32, 144.79, 135.95, 133.82, 132.02, 129.95, 128.04,127.69, 126.64, 123.12, 112.97, 85.89, 72.97, 70.64, 70.43, 69.97,69.74, 67.80, 64.34, 55.10, 37.14; Low resolution MS m/e calculated forC₄₀H₄₉O₁₀N₂ (M+NH₄ ⁺): 717.398, found 717.5. According to Scheme 3,compound 9 was synthesized as follows: To a stirred solution of 4a(10.13 g, 0.0279 mol) in 100 mL of acetone and 1 mL of H₂O was added3.98 g (1.22 equiv.) of N-methylmorpholine N-oxide. To this suspensionwas added 1.75 mL (0.005 equiv.) of Osmium tetroxide as a 2.5% solutionin iPrOH. After addition of the OsO₄ solution, the reaction mixturebecame clear yellow. After TLC analysis indicated complete conversion of4a (ca 16 h), the reaction mixture was treated with 1.5 g of sodiumhydrosulfite and 5.0 g of florisil and stirred 30 minutes. Thesuspension was filtered through florisil, the filtrate was concentratedto an oil. This crude product was combined with another batch preparedin the same manner from 1.0 g of 4a. Two 100 mL portions of pyridinewere co-evaporated from the combined lots and the residue was dissolvedin 300 mL pyridine. The solution was cooled to 0° C. and 10.89 g (1.05equiv.) of 4,4′-dimethoxytrityl chloride was added. A drying tube wasinserted in the flask and the reaction mixture was stirred at roomtemperature 16 h. The solution was treated with 20 mL of MeOH andconcentrated in vacuo, keeping the temperature of the water bath below40° C. The crude oil was purified by flash chromatography using 1100 mLof silica gel (wet-packed onto column using 3% triethylamine in hexane)and eluting with 10-100% EtOAc in hexane (all containing 3%triethylamine) to give 21.3 g (89% after two steps) of 9 as a yellowoil. ¹H NMR (300 MHz, CDCl₃) d 7.80-7.77 (m, 2H), 7.66-7.64 (m, 2H),7.39-7.22 (m, 9H), 7.20-6.76 (m, 4H), 3.97 (bs, 1H), 3.84 (t, J=5.97 Hz,2H), 3.74 (s, 6H), 3.68 (t, J=5.7 Hz, 2H), 3.60-3.49 (m, 14H), 3.13-2.76(m, 2H), 2.00 (bs, 1H); ¹³C NMR (75 MHz, CDCl3) d 168.2, 158.3, 144.8,135.9, 133.8, 132.0, 129.9, 128.0, 127.7, 126.6, 123.1, 113.0, 85.9,73.0, 70.6, 70.4, 70.0, 69.7, 67.8, 64.4, 55.1, 37.1; Low resolution MSm/e calculated for C₄₀H₄₅O₁₀N (M+NH₄ ⁺): 717.5.

1-Dimethoxytrityl-3-(aminotetraethylene glycolyl)-sn-glycerol (10):According to Scheme 2, compound 10 was synthesized as follows: Compound9 (5.2 g, 7.2 mmol) was taken up in 50 mL of 40% methylamine in H₂O and10 mL of methanol was added to solublize the starting material. Thereaction mixture was heated at 50° C. for 5 hr, and than wasconcentrated in vacuo and coevaporated with toluene. The crude materialwas purified by flash chromatography on 200 mL of silica gel, elutingwith 15% methanolic ammonia in dichloromethane. Collected 3.94 g (96%)of 10 as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) d 7.46-7.21 (m, 9H,DMT), 6.81 (d, 4H, DMT), 4.00 (m, 1H), 3.80 (s, 6H), 3.70-3.49(overlapping m, 18H), 3.20 (dd, J=9.24, 5.49 Hz, 1H), 3.12 (dd, J=9.21,6.0 Hz, 1H), 2.84-2.80 (m, 3H); ¹³C NMR (75 MHz, CDCl₃) d 158.30,144.82, 136.01, 129.95, 128.04, 127.66, 126.61, 112.95, 85.85, 73.46,72.85, 70.55, 70.45, 69.99, 69.51, 64.43, 55.10, 41.40; Low resolutionMS m/e calculated for C₃₂H₄₄O₈N (M+1⁺): 570.353, found 570.4.

According to Scheme 3, compound 10 was synthesized as follows: Compound9 (5.2 g, 7.2 mmol) was taken up in 50 mL of 40% methylamine in H₂O and10 mL of methanol was added to solublize the starting material. Thereaction mixture was heated at 50° C. for 5 hr, and than wasconcentrated in vacuo and coevaporated with toluene. The crude materialwas purified by flash chromatography on 200 mL of silica gel, elutingwith 15% methanolic ammonia in dichloromethane. Collected 3.94 g (96%)of 10 as a pale yellow oil. ¹H NMR (300 MHz, CDCl₃) d 7.46-7.21 (m, 9H,DMT), 6.81 (d, 4H, DMT), 4.00 (m, 1H), 3.80 (s, 6H), 3.70-3.49(overlapping m, 18H), 3.20 (dd, J=9.24, 5.49 Hz, 1H), 3.12 (dd, J=9.21,6.0 Hz, 1H), 2.84-2.80 (m, 3H); ¹³C NMR (75 MHz, CDCl₃) d 158.30,144.82, 136.01, 129.95, 128.04, 127.66, 126.61, 112.95, 85.85, 73.46,72.85, 70.55, 70.45, 69.99, 69.51, 64.43, 55.10, 41.40; Low resolutionMS m/e calculated for C₃₂H₄₄O₈N (M+1⁺): 570.353, found 570.4.

PEG Reagent 12: To a stirred solution of 0.24 g (0.41 mmol) of 10 in 10mL DMF was added 2.08 g (0.4 mmol) of methoxy-PEG₅₀₀₀-nitrophenylcarbonate (Shearwater Polymers, Inc.). The mixture was stirred 70 h thenconcentrated in vacuo. The residue was dissolved in EtOAc and theorganic phase washed with three 30 mL portions of 10% NaOH solution.Purification by flash chromatography using 100 mL of silica gel (wetpacked with dichloromethane containing 5% Et₃N) eluting with 5-10-15-20%methanolic ammnonia in dichloromethane afforded 1.97 g (85%) of 12 as awhite solid. ¹H NMR (300 MHz, CDCl₃) d 7.42-7.39 (d, 2H, DMT), 7.39-7.18(m, 7H, DMT), 6.79 (d, 4H, DMT), 5.70 (br m, 1H), 4.21 (m, 1H), 3.97 (m,1H), 3.88 (t, J=4.4 Hz, 1H), 3.81 (s, 6H, DMT), 3.78-3.50 (br m, ˜500,PEG Hs), 3.42-3.31 (overlapping ms), 3.35 (s, PEG Me), 3.16 (m, 2H),2.84 (br d, 4.2H); ¹³C NMR (75 MHz, CDCl₃) d 159.36, 157.19, 146.15,136.95, 130.83, 128.86, 128.65, 127.61, 113.86, 86.48, 73.58, 72.88,72.5-70.0 (PEG carbons), 68.31, 65.77, 64.45, 41.36, 30.75 (unassignedimpurity).

PEG Phosphoramidite Reagent 13: To a stirred solution of 2.22 g (0.4mmol) of 12 in 60 mL of THF over 3 A molecular sieves was added 0.24 mL(1.39 mmol) of diisopropylethylamine and 0.1 mL (0.44 mmol) of2-cyanoethyl N,N-diisopropylchlorophosphoramidite. After 5 h, 31 P NMRindicated formation of desired product, as well as hydrolyzedphosphitylating agent and an additional 0.05 mL (0.22 mmol) of2-cyanoethyl N,N-diisopropylchlorophosphoramidite was added. After 12 h,0.07 mL (0.4 mmol) of DIPEA and 0.1 mL of the chlorophosphoramidite wereadded. The mixture was stirred 2 days, filtered through Celite andconcentrated in vacuo. The residue was triturated with several portionsof ether. ³¹P NMR d 156.4, 155.8. Also obsered were signals at d 20.6,19.8 presumed to correspond to hydrolyzed phosphitylation reagent.

PEG H-Phosphonate Reagent 14: To a stirred, 0° C. solution of anhydrousimidazole (0.4 g; 5.9 mmol) in 10 mL of MeCN was added 0.1 7 mL (1.9mmol) of PCl₃, followed by 0.86 mL (6.2 mmol) of Et₃N. To this mixturewas added a solution of 3.0 g (0.55 mmol) of 12 in 12 mL of MeCNdropwise. The reaction mixture was allowed to warm to ambienttemperature and stirred 16 h. treated with 10 mL of 0.1 Mtriethylammonium bicarbonate solution, and concentrated in vacuo.Triethylamine, then toluene were coevaporated from the crude residue,then the product was dissolved in dichloromethane. The organic phase waswashed with 1.0 M triethylammonium bicarbonate (TEAB) solution, driedover sodium sulfate, and concentrated. Purification by flashchromatography on 300 mL of silica gel (wet packed with hexane/Et₃N(95:5)) eluting with 2-4-6-8-10-12-15% methanolic ammonia indichloromethane afforded 1.95 g of product as a white solid. ³¹P NMR(CDCl₃) d 10.3, 10.2.

Synthesis of Lipid phosphoramidite 17: A solution of 540 mg (1 mmol) of1,2-di-O-palmityl rac-glycerol in 10 mL of pyridine was treated with 195mg (1.2 mmol) of carbonyldiimidazole and the resulting mixture stirredat ambient temperature overnight. To this mixture was added 570 mg (1mmol) of the amino alcohol as a solution in 3 mL of DMF. The mixture waswarmed to 40° C. overnight, after which time ¹H NMR analysis of analiquot from the reaction indicated very negligible formation ofproduct. The mixture was heated to 80° C. for 6 h (ca 1:1 product tostarting material by ¹H NMR), then concentrated in vacuo. The cruderesidue was applied to a column of 125 mL of SiO2 gel (Packed inhexanes) and the product eluted with a gradient of 20-50% EtOAc inhexanes (with 2% TEA) to afford 500 mg (44%) of intermediate 16 as aclear wax. (16: ¹H NMR (300 MHz, CDCl₃) d 7.42 (d, J=7.2 Hz, 2H),7.30-7.18 (m, 7H), 6.76 (d, J=8.2 Hz, 4H), 5.34 (br t, 1H), 4.20-3.25(overlapping signals), 3.16 (m, 2H), 1.53 (m), 1.24 (m), 0.86 (t, J=6.5Hz, 6H). Alcohol 16 (500 mg; 0.44 mmol) was dissolved in 4 mL of CH₂Cl₂and 0.15 mL (2 equiv) of DIPEA. To this solution was added 0.15 mL (0.66mmol) of 2-cyanoethyl (N,N-diisopropylamino) chlorophosphoramidite.After 3 h, TLC showed conversion to 2 spots and the mixture was dilutedwith CH₂Cl₂ and washed with NaHCO₃ solution. The organic phase was driedover Na₂SO₄ and concentrated. The crude residue was applied to a columnof 50 mL of SiO₂ gel (packed in hexanes) and the product eluted with 20%EtOAc in hexanes (containing 2% TEA) to afford 350 mg (59%) ofphosphoramidite 17 as a colorless wax. ³¹P NMR (CDCl₃) d 151.55, 151.08.

Automated synthesis of lipid-oligo: Phosphoramidite 17 was coupled tothe 5′-end of a T-3mer (prepared by standard automated DNA synthesis onan ABI 394 instrument) using a modified coupling cycle consisting of two30 minute exposures of an 0.1 M solution of 17 in 40% THF in MeCN to thecolumn. In-line trityl analysis indicated a coupling efficiency of 94%for amidite 17.

Chloroformate 19: To a stirred solution of 3 g (5.03 nunol) of1,2-di-O-octadecyl-sn-glycerol 21 in 60 mL of toluene was added 20 mL ofa 1.93 M solution of phosgene. Additional phosgene solution (2×10 mL;15.4 equiv phosgene total) was added until no further alcohol startingmaterial remained (by ¹H NMR analysis of concentrated aliquots). Theexcess phosgene and HCI was removed by aspirator and the reactionmixture was concentrated in vacuo to afford 3.3 g (98%) of the desiredchloroforrnate 19 as a white powder. ¹H NMR (300 MHz, CDCl₃) d 4.45 (dd,J=11.22, 3.69 Hz, 1H), 4.34 (dd, J=1 1.22, 6.15 Hz, 1H), 3.65 (m, 1H),3.56-3.40 (m, 6H), 1.53 (m, 4H), 1.24 (m, 62H), 0.87 (t, J=6.36 Hz, 6H);¹³C NMR (75 MHz, CDCl₃) d 75.90, 71.91, 71.35, 70.93, 69.36, 31.99,29.96-29.44 (overlapping signals from hydrocarbon chains), 26.13, 26.04,22.76, 14.18.

Conjugate 20: To a stirred solution of 2.25 g (3.95 mmol) of 10 in 60 mLof pyridine was added 2.6 g of the distearyl glycerol chloroformate 18.¹H NMR analysis of a concentrated aliquot after 2 h revealed noremaining chloroformate and the mixture was concentrated in vacuo. Thecrude residue was combined with material similarly prepared from 0.5 g(0.88 mmol) of 10 and 0.58 g of the chloroformate and the combined lotspurified by flash silica gel chromatography on a column of 100 mL ofsilica gel (packed in hexanes containing 2% triethylamine) eluting with200 mL hexanes, then 250 mL each of 10-20 and 30% EtOAc in hexanes, 500mL 40% EtOAc in hexanes, then 250 mL each of 50-60-70 and 80% EtOAc inhexanes, and finally with 250 mL of EtOAc. The product containingfractions were concentrated to afford 3.3 g (57%) of the conjugate 20.

Phosphoramidite 21: To a stirred solution of 3.8 g (3.26 mmol) of theconjugate in 25 mL of CH₂Cl₂ was added 1.14 mL (6.52 mmol) ofdiisopropylethylamine then 1.09 mL (4.88 mmol) of 2-cyanoethylN,N-diisopropylchloro-phosphoramidite. After 2 hours, the mixture wasdiluted with CH₂Cl₂ and washed with saturated NaHCO₃ solution, driedover Na₂SO₄, and concentrated, The crude residue was purified by flashsilica gel chromatography on a column of 125 mL of silica gel (packed inhexanes containing 2% triethylamine) eluting with 100 mL hexanes, then250 mL each of 10 and 20% EtOAc in hexanes, 500 mL 30% EtOAc in hexanes,then 250 mL of 50% EtOAc in hexanes. The product containing fractionswere concentrated to afford 4.2 g (95%) of the phosphoramidite 21. ³¹PNMR (CDCl₃) d 151.52, 151.08.

EXAMPLE 2

Preparation and Functional Properties of PEG Conjugated andCholesterol-Derivatized Nucleic Acid Ligands

A PEG 3400 Conjugate of a Nucleic Acid Ligand Retains the BindingAffinity of the Non-Conjugated Molecule

The ability of a bFGF ligand/PEG-3400 conjugate to bind bFGF was tested.Molecule 225t3 (SEQ ID NO: 10), a high affinity DNA ligand to bFGF, waschosen for conjugation with PEG via a primary amine-NHS couplingreaction. Ligand 225t3 has a binding affinity of 1 nM and folds into ablunt ended hairpin with a Tm of 68° C. Ligand 225t3 was modified with a3′-amino-modifier C7 CPG (Glen Research, Sterling, Va.) using standardDNA synthesis methods and will be referred to as 225t3N (SEQ ID NO:11).225t3N was reacted with the N-hydroxysuccinimidyl (NHS) active ester ofPEG (avg. MW 3400) in 20% (v/v) dimethoxy formamide 80% (v/v) 0.5 Msodium bicarbonate buffered at pH 8.5. The resulting conjugate,225t3N-PEG-3400 (SEQ ID NO:14), was purified from the free DNA on a 12%polyacrylimide/7 M urea gel. The conjugate was 5′ end-labeled with ³²Pand a binding assay was performed. 225t3N-PEG-3400 (SEQ ID NO:14) bindsto bFGF with the same affinity (K_(d)=1 nM) as 225t3.

Conjugation of PEG-20,000 to a Thrombin DNA Ligand.

Thrombin DNA ligand NX256 (SEQ ID NO:9) (FIG. 1D) containing an aminoand a disulfide functionality was prepared using standard DNA synthesismethods and procedures on a Biosearch 8909 DNA/RNA synthesizer withdT-5′-LCAA-500 Å controlled-pore glass solid support and commerciallyavailable phosphoramidite reagents. Deprotection was followed by ionexchange HPLC purification. The 5′ terminal disulfide bond was reducedby incubating the DNA in a solution 50 mM dithiolthreitol (DTT) at 37°C. for 30 min. The reduced DNA (containing a 5′ terminal thiol) was runthrough a Nap-5 size exclusion column and the void volume, containingthe DNA but not the DTT, was collected into a reaction vessel containingthe maleimide derivatized PEG under an argon blanket. All solutions werepurged with argon to remove oxygen. The reaction was kept at 40° C. for1 h. The progress of the reaction was monitored by removing smallaliquots and analyzing them by electrophoresis on 8%/7 M ureapolyacrylamide gels. At the end of the 1 hour incubation period, thereaction was essentially complete, and at this time an equal volume ofmethylene chloride was added to the reaction mix and the vessel shakenuntil a milky white suspension formed. The mix was spun at 14,000 rpm inan eppendorf centrifuge until the layers separated. The aqueous layercontained the free Nucleic Acid Ligand and was discarded. The product(PEG-20,000 modified DNA ligand NX256, referred to as NX256-PEG-20,000;SEQ ID NO: 13) (FIG. 1G) was further purified using ion exchangechromatography followed by reverse phase desalting and lyophilization toa white powder. This material was used to determine the effect of PEGmodification on the pharmacokinetic behavior of the DNA Nucleic AcidLigand (vide infra). PEG-10,000 modified ligand NX256, referred to asNX256-PEG-10,000, was prepared in an analogous manner.

T_(m) Values for PEG Conjugates

PEG functionality can also be introduced via the thiophosphate-maleimidereaction. We introduced the thiophosphate group into the thrombin DNAligand at the 5′-end by the standard phosphoramidite method usingcommercially available reagents (this ligand is referred to as T-P4)(FIG. 1F, SEQ ID NO:12). The conjugation to the maleimide-containing PEGis analogous to the sulfhydryl-maleimide reaction described above. ThePEG-conjugated ligands are referred to as T-P4-PEG-10,000 andT-P4-PEG-20,000 (SEQ ID NO:15). Melting temperatures (T_(m)) forT-P4-PEG-10,000, T-P4-PEG-20,000 and T-P4-DNA ligands were determinedfrom the first derivative of the optical absorbance at 260 nm vs.temperature plots. The T_(m) values for T-P6-PEG-10,000, T-P6-PEG-20,000and T-P6 were 40° C. for all three ligands. These data and thebFGF-PEG-3400 Nucleic Acid Ligand binding data reported above, suggestthat conjugation to PEG does not affect Nucleic Acid Ligand structure.

A Cholesteylated bFGF Ligand Retains the Binding Affinity of theNon-Cholesterylated Molecule

Cholesterol can be introduced into a Nucleic Acid Ligand, at anyposition in the sequence, by the standard solid phase phosphoramiditemethod. For example, we incorporated a tetraethyleneglycol cholesterolphosphoramidite (Glen Research, Sterling, Va.) at the 3′ end of ligand225t3 (FIG. 1E; SEQ ID NO: 10) to produce ligand 225t3-CholesterolFollowing purification on a 12% polyacrylimide/7 M urea gel, 225t3-Cholwas 5′ end-labeled with ³²P and a binding assay performed. The bindingaffinity of 225t3-Chol was identical (K_(d)=1 nM) to that of 225t3.

EXAMPLE 3

Nucleic Acid Ligand-Liposome Formulation and Anticoagulation Activity

A. Preparation of NX232 Liposomes

Fluorescein-labeled, cholesterol-derivatized NX232 (FIG. 1B; SEQ IDNO:7) was incorporated into Liposomes composed primarily ofdistearoylphosphatidylcholine (DSPC) and cholesterol (Chol) in a 2:1molar ratio.

Eight formulations of NX232 Liposomes containing DSPC:Chol (2:1 moleratio) were prepared. The mole percentage of NX232 was varied from 0.01through 0.1 mole %, based upon total lipids present. The compositions(A-H) are reported in Table 1. Increasing fractions of the cationiclipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were included informulations D-H to evaluate the effect of positive charges on thestrength of the association between NX232 and the Liposome surface.TABLE 1 Summary of Liposome NX232 Preparations - Lipid compositions andmole percentage of NX232 Compound^(a) M.W. A B C D E F G H molefractions: DSPC 790.15 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Chol 386.7 1.01.0 1.0 1.0 1.0 1.0 1.0 1.0 DOTAP 698.55 0.0 0.0 0.0 0.0 0.0012 0.0030.006 0.012 NX232 12,424.1 0.0003 0.0008 0.0015 0.003 0.0003 0.00080.0015 0.003 (NX232 mole-%:) 0.01 0.025 0.05 0.1 0.01 0.025 0.05 0.1Weight Ratios: DSPC 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Chol 0.2447 0.24470.2447 0.2447 0.2447 0.2447 0.2447 0.2447 DOTAP 0.0 0.0 0.0 0.0 0.00050.0013 0.0027 0.0053 NX232 0.0024 0.0059 0.0118 0.0236 0.0024 0.00590.0118 0.0236 Amounts per ml^(b): DSPC 20.0 20.0 20.0 20.0 20.0 20.020.0 20.0 Chol 4.894 4.894 4.894 4.894 4.894 4.894 4.894 4.894 DOTAP 0.00.0 0.0 0.0 0.011 0.027 0.053 0.106 NX232 0.047 0.118 0.236 0.472 0.0470.118 0.236 0.472^(a)DSPC = distearoylphosphatidylcholine; Chol = cholesterol; DOTAP =1,2-dioleoyl-3-trimethylammonium-propoane.^(b)final total concentration for all components = 25 mg/ml.

Lipids and NX232 were co-solublized in a mixture of chloroform, methanoland water (CHCl₃:MeOH:H₂O, 1:5:1, v:v:v) and transferred to test tubes.Lipid films were formed by evaporating the solvent under nitrogen flow.The dried lipid films were stored under vacuum until hydration. Thefilms were hydrated at 65° C. with an aqueous 9% sucrose solution (˜250mM) containing 10 mM tris(hydroxymethyl)aminomethane (TRIS) and 1 mMethylenediaminetetraacetic acid (EDTA) at pH 7.4. Following hydration,the lipids were sonicated using a probe-type sonicator for approximately6-9 minutes and then cooled to room temperature.

Unincorporated NX232 (FIG. 1B; SEQ ID NO:7) was removed by FPLC gelpermeation chromatography using Sephacryl S-300, eluting with thesucrose hydrating solution described above. The gel permeationchromatograms for empty Liposomes (Empty), Liposomes with 4.7 μg NX232(L-NeX2a), Liposomes with 11.8 μg NX232 (L-NeX2b), free NX232 at 72 μg(Free 72 μg), free NX232 at 7.2 μg (Free 7.2 μg), and free NX232 at 10μg which had been sonicated (Free/soni 10 μg) are shown in FIG. 2. Thechromatograms indicate that good separation of free NX232 and Liposomesis possible. The free NX232 chromatogram at 72 μg shows a distinct peakfollowing the Liposomes (elution peak at ˜53 minutes); however, 72 μg ofthe free Nucleic Acid Ligand had to be added in order to be visualizedby UV absorbance at 254 nm. No similar peak can be discerned for freeNX232 at 7.2 μg. Therefore, with the liposomal preparations, the NX232amounts (4.7 μg for “a” and 11.8 μg for “b”) may have been insufficientto show up as a distinct peak on the chromatogram.

Based on the absorbance measurements at 254 nm, NX232 is incorporatednearly quantitatively into these Liposomes. Since only approximately 50%of the NX232 molecules were fluorescein labeled, quantitativeincorporation suggests that the presence of the fluorescein label doesnot significantly affect association of NX232 with Liposomes.

B. In Vitro Assay of Clotting Inhibition

NX232 (FIG. 1B; SEQ ID NO:7) contains the DNA sequence of a highaffinity Nucleic Acid Ligand to thrombin. Thrombin is a criticalcomponent of the blood clotting cascade. The inhibition of theproteolytic activity of thrombin is known to decrease the ability ofblood to clot. The activity of the various NX232 formulations wereevaluated using a fibrin/thrombin clotting assay to measureanticoagulation activity. A buffer solution of 50 mM TRIS, 100 mM NaCl,1 mM MgCl₂, and 0.1% polyethylene glycol (PEG₈₀₀₀) (MW 8,000) at pH 7.4was used for the assay. In the final 300 μl assay mixture, fibrinogen ata concentration of 2.5 mg/ml and thrombin at 1 National Institutes ofHealth (NIH) unit were added to glass test tubes. All solutions andcontainers were warmed to and maintained at 37° C. for coagulationmeasurements. Coagulation times are reported in Table 2. TABLE 2 Effectof Processing on NX232 Anticoagulation Activity NX232 Clot TimePreparation (μg/300 μl) (sec.) Control (no additions) — 18-20 NX232,unsonicated 7.08 49-51 3.54 19-21 NX232, sonicated 7.08 45-50 3.54 20-25Liposomes: Preparation A 7.08 57-59 3.54 26-28 Preparation B 7.08 56-593.54 25-27

These results indicate that typical Liposome processing conditions,including sonication, solubilization, heating and drying, do not affectthe anticoagulation activity of NX232. In addition, liposomalassociation does not affect the ability of NX232 to bind and inhibit itstarget.

Example 4

Pharmacokinetic Properites of Cholesterol, Diacyl Glycerol, DialkylGlycerol, and PEG Modified DNAS.

The pharmacokinetic properties of thrombin DNA ligands NX229, NX232,NX253, NX253+Liposome, and NX256-PEG20K were determined (see FIGS.1A-1C, 1G for molecular descriptions) (SEQ ID NOS:6-8, 13). Eacholigonucleotide was diluted in PBS to a solution concentration of0.5-1.0 mg/ml based on UV absorption at 260 nm and an extinctioncoefficient of 0.033 μg oligo/ml. In all but one study, 6 rats received0.5-1.0 mg oligonucleotide/kg animal weight and plasma samples weretaken at various times from 1 minute to 4 hours. One rat was used in thestudy in which NX253 was tested. The plasma samples and quality controlsamples were analyzed using a hybridization assay. The hybridizationassay utilized a capture oligonucleotide that contains a complementarysequence to the 5′-end of the DNA ligands conjugated to an iron oxide(FeO) bead (FeO-spacer-5′-d (GTC AGG CAC CAT CCC-3′) (SEQ ID NO: 1)where spacer=(dT)₈), and a detection oligonucleotide containing a biotingroup at the 3′-end (5′d-CCC CAC TGA AGC ACC-spacer-3′-biotin-biotin,where spacer=(dT)₁₀) (SEQ ID NO:2). The amount of the biotinoligonucleotide attached to the bead was quantitated with thestraptavidin-linked alkaline phosphatase, using CSPD-Sapphire as theluminescent substrate.

Data for the plasma concentration of NX229, NX232, NX253,NX253+Liposome, and NX256-PEG20K (SEQ ID NOS:6-8, 13) as a function oftime following the bolus injection are summarized in FIG. 3. The plasmaconcentrations of NX232, NX253, NX253+Liposome, and NX256-PEG20K as afunction of time are considerably greater compared to that of NX229 (SEQID NO:6). All of these oligonucleotides share the same thrombin bindingmodule (d(CAG TCC GTG GTA GGG CAG GTT GGG GTG ACT TCG TGG)) (SEQ IDNO:3). The plasma concentration of an oligonucleotide as a function oftime can be significantly increased by introducing appropriatefunctional groups into the oligonucleotide. Prolonged plasma half-lifeof a cholesterol-containing oligonucleotide compared to the control(non-cholesterol-containing) oligonucleotide has been observedpreviously (de Smidt et al., Nucl. Acids Res., 19: 4695 (1991))

The plasma pharmacokinetic properties of a wide number of Nucleic AcidLigands that have various functional groups attached to the basesequence of NX213 (see FIG. 1 for molecular description), as well assome liposomal formulations of these oligonucleotides have beenassessed. These data are summarized in FIG. 4 (SEQ ID NOS:16, 19, 21, 22and 29). The formulation with the slowest clearance rate was where NX213(FIG. 1P; SEQ ID NO:21) was encapsulated within liposomes.

To determine the role of dialkyl glycerol DNA conjugates plus and minusliposomes PK studies 77 and 73 were carried out with the Thrombin DNAligand dialkyl glycerol conjugate (See FIG. 1 for moleculardescription). These data are summarized in FIG. 5 (SEQ ID NO: 18).

Cholesterylated VEGF oligonucleotides NX213 (NX268) (See FIG. 1K formolecular description; SEQ ID NO: 16) were formulated with eitherPEG-liposomes or standard liposomes and the pharmacokinetics wereevaluated (PK 85-86). These formulations contain oligonucleotides bothon the inside and outside of the liposome. FIG. 6 (SEQ ID NO: 16) showsthe rat plasma levels of full length oligonucleotides as a function oftime after injection. Both liposome formulations show similaroligonucleotide pharmnacokinetics.

To evaluate size dependence on clearance, 2′ O-methyl VEGFoligonucleotides with various PEG conjugates (See FIG. 1 for moleculardescription) (PK 50,80,87, &88) have been studied. FIG. 7 shows thecomparison of the all 2′ O-methyl oligonucleotides plus PEG 40K, 20K,10K, as well as in the absence PEG (SEQ ID NOS: 17 and 29). These datademonstrate significantly slower plasma clearance with increasing sizeof the PEG conjugate.

PK 96 was carried out to evaluate the pharmacokinetics of 2′ Fpyrimidines in conjunction with 2′ O-methyl purines and PEG20K (JW1130)(See FIG. 1R for molecular description; SEQ ID NO:23). FIG. 8 shows theplasma levels of this oligonucleotide in comparison with the all 2′O-methyl version (PK 80, JW986) (See FIG. 1X for molecular description;SEQ ID NO:29). These data show fairly similar clearance properties forboth oligonucleotides.

The observation that JW1130 (SEQ ID NO:23), containing 2′ F pyrimidines,shows similar clearance to JW986 (2′ O-methyl pyrimidines) suggests thatoligonucleotides with 2′ F pyrimidines are resistant to nucleasedigestion.

PK 97 was carried out to determine the clearance properties of aL-Selectin DNA ligand (NX287) (FIG. 1S; SEQ ID NO:24) conjugated with40K PEG. FIG. 9 shows the plasma levels of this oligonucleotide as afunction of time after bolus injection (dose 1 mg/kg). For comparison,Thrombin DNA ligand (NX256) (FIG. 1H; SEQ ID NO: 13) conjugated with 20KPEG was included. As shown in FIG. 9 these two oligonucleotides showsimilar clearance rates presumably due to metabolism.

PK studies 99, 100 and 102 have been carried out as part of a largerstudy to assess the stability of oligonucleotides in vivo. These studiesare shown in FIG. 10. For comparison, PK 96 (JW1130 VEGF) 2′F Py 2′O-Met(14 Pu)+PEG 20K ) (FIG. 1R; SEQ ID NO:23) is also included. Theoligonucleotides used in PK studies 96, 100, and 102 differ only in thenumber of purine positions that contain 2′ deoxy nucleotides, where PK96 contains no 2′ deoxy purines, PK 100 four 2′ deoxy purines, and in PK102 all 14 purines are 2′ deoxy. FIG. 10 (SEQ ID NOS:23, 25, 26. 27 and30) demonstrates a clear relationship between increasing clearance rateand the number of deoxy nucleotides present in the oligonucleotide. Thisobserved increase in clearance rate with increasing number of deoxynucleotides is assumed to be due to increased metabolism of theseoligonucleotides. An encouraging observation is the high level ofstability shown for PK 100 containing four 2′ deoxy purines, andsuggests that post-SELEX modification may be appropriate if a largenumber of purines can be modified. Also shown in FIG. 10 is PK 99 vs. PK100, that differ in PEG20K conjugation. As previously observed withother oligonucleotides, conjugation to PEG molecules of significantmolecular weight dramatically reduces the observed clearance rate fromplasma.

EXAMPLE 5

Cationic Liposome-Nucleic Acid Ligand Complexes: Toxicity and TheirIntracellular Uptake by Human Lymphocytes.

Toxicity. To determine toxic effects of Liposome-Nucleic Acid Ligands oncells, the human primary peripheral blood lymphocytes (PBLs) weretreated with Nucleic Acid Ligand alone, the two types of Liposomesalone, and the two Liposome-Nucleic Acid Ligand combinations (videinfra). Two different types of Liposomes, type 1(=dioleoylphosphatidylethanolamine (DOPE):aminomannose cholesterol at1:1 weight ratio) and type 2 (=DOPE:aminomannose cholesterol:DOTAP at a1:1.5:1 weight ratio) were used for this study. The Liposomes were mixedat a ratio of 5:1 (Liposome:Nucleic Acid Ligand, by weight) with singlestranded DNA SELEX ligand RT1t49-PS (5′-d(ATC CGC CTG ATT AGC GAT ACTCAG AAG GAT AAA CTG TCC AGA ACT TGG AsTsTsTsT)-3′ (SEQ ID NO:4), wherelowercase s indicates a phosphorothioate linkage) that binds to HIV-1reverse transcriptase with a K_(d) of approximately 1-5 nM. The CationicLiposome-Nucleic Acid Ligand Complex was formed by incubating theLiposome and the type 1 or type 2 Liposome with RT1t49-PS at 65° C. for10 minutes.

PBLs (phytohemagglutinin and natural IL-2 stimulated) were plated at adensity of 2×10⁵ cells per well in 96-well plates. Cells are treated atday 0, split and retreated at day 4 with Nucleic Acid Ligand alone, orthe two types of Liposomes alone, or the two Liposome-Nucleic AcidLigand Complexes. The viable cells were counted at day 7. The percent ofviable cells for each of the treatment groups is summarized below:Treatment % Viability cells alone 85 30 μg/ml RT1t49PS 82 Liposome type1 (150 μg/ml) 85 Liposome type 2 (150 μg/ml) 63 Liposome type 1 (150μg/ml) + RT1t49PS (30 μg/ml) 88 Liposome type 2 (150 μg/ml) + RT1t49PS(30 μg/ml) 77

These results suggest the following. Ligand RT1t49PS was not toxic atconcentrations up to 30 μg/ml. Liposome type 1 not toxic atconcentrations up to 150 μg/ml while Liposome type 2 is moderately toxic(about 25% reduced viability) at this concentration. The toxicity ofLiposome type 2 is expected because DOTAP is known to be toxic. LigandRT1t49PS apparently reduces the toxicity of Liposome type 2 by about˜50%.

Cellular uptake. Intracellular delivery of fluoresceinated RT1t49PSligand was examined with CEMss cells (human T cell line) usingfluorescence activated cell sorting (FACS) analysis and confocalmicroscopy. For this study DOPE:aminomannose (1:1 mole ratio) Liposomeswere used. Lipid films were prepared from 2.33 mg DOPE and 2.33 mgaminomannose, dissolved in chloroform, and kept under vacuum in adesiccator overnight. 1 ml of 9% sucrose was added to the film and thetube was heated at 65° C. for 0.5 minutes and vortexed. The lipidmixture was then sonicated at a power setting of 7 (microtip) for 2minutes in a beaker containing water heated to 50° C. An additional 0.5ml of 9% sucrose was added to the Liposomes and the Liposomes were sizedusing a MicroTrac particle sizer (average size 45 nm) and sterilefiltered using a 0.45 μm cellulose acetate filter. Liposome-Nucleic AcidLigand Complexes were prepared by incubating the Liposome with theNucleic Acid Ligand at a 5:1 lipid:oligonucleotide w/w ratio for 10minutes at 65° C.

For the cellular uptake experiment, 10⁶ CEM cells were diluted in 10 mlof 1640 RPMI/10% fetal calf serum in T25 flasks. The Nucleic Acid Ligandat 0.6 μM, as free drug or in a Liposome Complex, was added to eachflask and incubated at 37° C. in an atmosphere containing 5% CO₂. Priorto observation, cells were centrifuged and washed twice to remove excessdrug.

Confocal Microscopy was performed with an air-cooled argon laser(excitation 488 nm). Confocal images were taken at 1 μm slices(approximately 20 slices per series). Through 24 hours, no significantfluorescence (above background) was detectable in CEM cells incubatedwith Nucleic Acid Ligand alone. Significant fluorescence was detected inCEM cells incubated with Liposome-associated Nucleic Acid Ligands by 5hours and increased through 24 hours. Fluorescence appeared to belocalized in small vacuoles and not in the nucleus. In polarized cells(example 7 hr incubation with Liposome-associated Nucleic Acid Ligand),the fluorescence is localized in the rear of the cell away from theleading/advancing edge.

FACS analyses were performed with a Coulter Epics Elite equipped with anair-cooled argon laser (excitation 488 nm). CEM cells were gated forforward and side scatter and examined for green fluorescence. Dead cellsand aggregates were excluded from the gate. As suggested by confocalmicroscopy, the fluorescence of cells incubated with Liposome-associatedNucleic Acid Ligand is about an order of magnitude greater than that ofcells incubated with free Nucleic Acid Ligand. Uptake ofLiposome-associated Nucleic Acid Ligand is not entirely homogeneous.Some cells are significantly more fluorescent than others. For a 5:1 w/wlipid:Nucleic Acid Ligand ratio (M_(r) of Nucleic Acid Ligand≈14,0000;M_(r) of lipid≈700) and assuming 40,000 lipids per Liposomes, there areapproximately 400 Nucleic Acid Ligands per Liposome. The lower detectionlimit of the FACS is approximately 500 fluorophores per cell or slightlygreater than 1 Liposome per cell.

In conclusion, free 5′F1-RT1t49PS Nucleic Acid Ligands do notsignificantly localize within CEM cells within 24 hours. Nucleic AcidLigands associated with DOPE:aminomannose Liposomes localize within CEMcells by 5 hours and continue to localize in the cells through 24 hours.Liposome-associated Nucleic Acid Ligands appear to accumulate invacuoles and not in the nucleus. The amount of Liposome-associatedNucleic Acid Ligand uptake is at least ten times greater than for freeNucleic Acid Ligand, as judged by FACS analysis.

EXAMPLE 6

Incorporation of Nucleic Acid Ligands Into Preformed Liposomes: Effectof Varying the Negative Charge of the Lipids.

Small unilamellar vesicles (SUV) composed ofdistearoylphosphatidylcholine DSPC), cholesterol (Chol), anddistearoylphosphatidylglycerol (DSPG) were prepared using formulationswith the molar ratios shown in Table 3. Four compositions containingvarying molar percentages of DSPG, a negatively-charged lipid, wereprepared to evaluate the effect of negative Liposome charge on theincorporation of a polyanionic Nucleic Acid Ligand. The lipids weredissolved in CHCl₃, mixed and dried under a steady stream of nitrogen.The dried lipid film was further dried and stored under vacuum overnightprior to hydration. The lipid film was hydrated with a pH 7.4 phosphatebuffer solution (PBS), containing Na₂HPO₄ (1.15 g/L), NaH₂PO₄ (0.228g/L), and sucrose (90 g/L), at 65° C. to yield a 50 mg/mL lipidsuspension. The hydrated lipid suspension was then sonicated for 15-30min using a probe-type sonicator until an opalescent solution wasobtained.

These preformed SUV were added to an equal volume of Nucleic Acid Ligand232 (NX232) (SEQ ID NO:7), 1.0 mg/mL in PBS (final concentrations: 0.5mg/mL NX232, 25 mg/mL lipid). The mixture was incubated at 65° C. for 15min or kept at room temperature before being chromatographed on aSephacryl HR S300 size-exclusion column (0.5×20 cm) to separate freefrom SUV-bound NX232. Chromatography conditions were as follows: eluent,PBS described above; flow rate, 0.1 mL/min; sample injected, 25 μL;detector, UV absorbance at 254 nm; fraction, 0.2 mL/fraction. Thecollected fractions were also monitored by fluorescence intensity(excited at 494 nm and emitted at 516 nm).

SUV-associated NX232 eluted with the SUV peak (excluded volume) andfree-NX232 eluted in the included volume. The chromatogram (FIG. 4)clearly demonstrates that the degree of NX232 association with SUV wasdependent upon the DSPG content in the SUV. As the percentage ofnegatively charged DSPG contained in the SUV was increased betweensamples A-D, NX232 association with SUV decreased. TABLE 3 Compositionof Liposomes with Various Negative Charges Molar Percentage Lipid (A)(B) (C) (D) DSPC 85 87.5 89 90 Cholesterol 10 10 10 10 DSPG 5 2.5 1 0

EXAMPLE 7

Incorporation of Nucleic Acid Ligands Into Preformed Liposomes: Effectof Varying the Cholesterol Content

SUV composed of DSPC and Chol were prepared and NX232 incorporationassayed as in Example 6. The Liposomes contained different molar ratiosof DSPC and cholesterol as indicated in Table 4. NX232 associated withthe Liposomes and eluted with the SUV when prepared at room temperature.TABLE 4 Composition of Liposomes with Various Cholesterol Contents Mole% Mole % Formulation DSPC Cholesterol (E) 95 5 (F) 90 10 (G) 85 15 (H)80 20 (I) 75 25 (J) 70 30 (K) 65 35

Liposome formulations J and K (approximately 2:1 mole ratio ofDSPC:cholesterol) allow for the most efficient incorporation of theNucleic Acid Ligent NX232.

EXAMPLE 8

Incorporation of Nucleic Acid Ligands Into Preformed Liposomes: Effectof Varying Lipid/Nucleic Acid Ligand Ratio with a Fixed Amount of NX232

DSPC:Chol (2:1 molar ratio) SUV were prepared and assayed as in Example6, but with varying lipid/NX232 ratios. A fixed amount of NX232 (SEQ IDNO:7), 1.0 mg/mL, was mixed at room temperature with an equal volume ofSUV, containing lipid concentrations from 2.5 to 50 mg/mL (Table 5). Theresults suggest that the maximal association of NX232 with SUV wasachieved at lipid/NX ratios (w/w) of 25/1. The highest lipid/NX ratio,50/1, did not increase the amount of NX232 bound to SUV. TABLE 5Lipid/NX Ratios (w/w) Tested with A Fixed Amount of NX232 Lipid Conc. NXConc. Lipid/NX (mg/mL) (mg/mL) Ratio (w/w) 2.5 1.0 2.5/1  5.0 1.0  5/110.0 1.0 10/1 25.0 1.0 25/1 50.0 1.0 50/1

EXAMPLE 9

Incorporation of Nucleic Acid Ligands Into Preformed Liposomes: Effectof Varying Lipid/Nucleic Acid Ligand Ratio with a Fixed Amount of SUV

Preformed SUV (DSPC/CH:2/1), 50 mg/mL, prepared as in Example 6, weremixed with an equal volume of NX232 at various concentrations from 0.5to 5.0 mg/mL at room temperature (Table 6). The results indicate thatmaximal association of NX232 with SUV was obtained at a lipid/NX232ratio (w/w) of 25/1. The fraction of NX232 associated with SUV decreasedwith lower lipid/NX232 ratio. TABLE 6 Lipid/NX Ratios (w/w) Tested withA Fixed Amount of SUV Lipid Conc. NX Conc. Lipid/NX (mg/mL) (mg/mL)Ratio (w/w) 50 5.0 10/1 50 4.0 12.5/1   50 3.0 16.7/1   50 2.5 20/1 502.0 25/1 50 1.0 50/1 50 0.5 100/1 

EXAMPLE 10

Incorporation of Nucleic Acid Ligands Into Preformed Liposomes: Effectsof Varying the Phospholipid Chain Length

SUV were prepared as in Example 6 from the phospholipids indicated inTable 7 to study NX232 association with SUV made of phospholipids withdifferent chain length. TABLE 7 Composition of Liposomes with VariousPhospholipids Formulation Phospholipid/Cholesterol Molar Ratio (M)Distearoylphosphatidylcholine (C18)/Chol 70/30 (N)Dipalmitoylphosphatidylcholine (C16)/Chol 70/30 (O)Dimyristoylphosphatidylcholine (C14)/Chol 70/30

Of the three formulations tested, the DPPC/cholesterol Liposome appearsto have the highest capacity to incorporate the Nucleic Acid LigandNX232.

EXAMPLE 11

Analysis of the Nucleic Acid Ligand-Liposome Complex by Non-DenaturingGel Electrophoresis.

In this example, the incorporation of a Nucleic Acid Ligand conjugatewith cholesterol into the liposomal formulation is demonstrated. TheLiposome formulation used in this study is the DSPC:Cholesterol (2:1,mol/mol). The ability of the Liposome to incorporate the cholesterylatedthrombin ligand NX253, radiolabeled with ¹²⁵I-Bolton-Hunter reagent(5′-[Cholesterol][dT-NH-¹²⁵I-Bolton-Hunter]-d(CAG TCC GTG GTA GGG CAGGTT GGG GTG ACT TCG TGG AA)[3′3′dT]dT-3′ (SEQ ID NO:8). wheredT-NH-¹²⁵I-Bolton-Hunter is the Amino-Modifier C6 dT (Glen Research,Sterling, Va.) conjugated to the Bolton-Hunter reagent (New EnglandNuclear, Boston, Mass.) and 3′3′dT (dT-5′-CE phosphoramidite, GlenResearch, Sterling, Va.) is the inverted-orientation phosphoramidite)was examined by determining the fraction of bound Nucleic Acid Ligand asa function of Liposome:Nucleic Acid Ligand ratio. The Nucleic AcidLigand-Liposome Complexes were prepared by incubating the Nucleic AcidLigand with the Liposome in 25 mM Tris buffer, pH 7.4 containing 9%sucrose at 65° C. for 15 min. The free Nucleic Acid Ligand can beseparated from the Liposome-bound Nucleic Acid Ligand by non-denaturingpolyacrylamide gel electrophoresis. This method allows for rapid andcomplete separation of the two species. In order to allow theLiposome-bound Nucleic Acid Ligand to enter the gel (so that it can bevisualized), it is necessary to disrupt the Liposomes by adding a 1%solution of triton X-100 to the loading wells for about 5 minutes priorto termination of the electrophoresis run. The amount of Liposome-boundNucleic Acid Ligand was determined from the relative amount of the freeNucleic Acid Ligand, which runs as a well-defined band, byphosphorimager analysis. Assuming that there are approximately 60,000lipids per Liposome, and that the mean MW of a lipid is 655.9 Da(=790.15×0.67+386.7×0.33), the saturation of the Liposome with theNucleic Acid Ligand occurs at the molar ratio of Nucleic Acid Ligand toLiposome of approximately 300 (FIG. 12). The analog of NX253 that doesnot have the cholesterol moiety is not incorporated into the Liposomeover the same range of Liposome concentrations (data not shown).

EXAMPLE 12

Passive Encapsulation of Nucleic Acid Ligands Into Liposomes

The Nucleic Acid Ligands are encapsulated within the aqueous interior ofLiposomes. An aqueous solution of a Nucleic Acid Ligand is prepared bydissolving the Nucleic Acid Ligands in phosphate buffer solution (PBS)to yield a stock solution with a concentration of approximately 3.5mg/ml. A lipid film containing DSPC:Chol (2:1 mole ratio) is prepared bydrying the lipid mixture from chloroform:methanol:water (1:5:1, v:v:v)solvent. One ml of the Nucleic Acid stock solution is added to the lipidfilm and bath sonicated at a temperature of 40° C. for 10 seconds. Theresulting solution is put through a 4-cycle freeze-thaw procedure usingliquid nitrogen. The resulting homogeneous solution is extruded firstthrough a 0.8 μm filter membrane (3 times) then extruded through a 0.45μm filter (3 times) and finally through a 0.2 μm filter (3 times).Unencapsulated Nucleic Acid Ligands are removed by passing thedispersion through a Sephadex G-50 column with a bed volume of about 20ml.

EXAMPLE 13

Remote Loading of Nucleic Acid Ligands Into Liposomes

The Nucleic Acid Ligands are encapsulated within the aqueous interior ofMLVs by remote loading. A lipid mixture of DSPC:Chol (2:1 mole ratio) isprepared as a lipid film using 20 μmol of lipid. The lipid film isvortexed into suspension using 0.1 M MgCl₂ at 65° C. to form MLVs havingan average diameter of one micron. The Liposome suspension is frozen inliquid nitrogen and thawed at 65° C. The freeze/thaw cycling is repeatedthree times to ensure that the salt is uniformly distributed throughoutthe lamellae. The osmolarity of the internal aqueous phase isapproximately 300 milliosmoles (mOsm). The Liposome suspension ispelleted by centrifugation at 10,000 g for 15 minutes to remove externalMgCl₂ solution. The supernatant is removed and the Liposome pellet isheated at 65° C. for 5 minutes. A solution of Nucleic Acid Ligand (20 μgin 100 μl water) is preheated for 5 minutes at 65° C. and added to theLiposome pellet. Heating is continued for 30 minutes and the sample isthen slowly cooled to room temperature and diluted with 1 ml PBS.Unentrapped Nucleic Acid Ligand is removed by centrifugation of the MLVsfollowed by supernatant removal. The pellet is resuspended in fresh PBSand re-pelleted by centrifugation.

EXAMPLE 14

Covalent Conjugation of Nucleic Acid Ligands to Liposomes

In scheme 1 provided below, a heterobifunctional PEG-2000 (PEG withmolecular weight 2000 Da) containing a N-hydroxysuccinimide ester andvinyl sulfone functionalities was first conjugated to a Liposomecontaining 2 mole % distearylphosphatidylethanolamine (DSPE) via theN-hydroxysuccinimide ester moiety. The product was purified from thefree PEG by size exclusion chromatography. The vinyl sulfone product wasthen allowed to react with reduced NX256 (FIG. 1D; SEQ ID NO:9). ADSPE-PEG-2000-vinyl sulfone is commercially available and can be used tomanufacture Liposomes that contain the vinyl sulfone functionality, thuseliminating a conjugation step from Scheme 1.

The second reaction, shown below as scheme 2, has been completed. Thestarting material was a distearylphosphatidylcholine (DSPC) Liposomecontaining 2 mole % DSPE maleimide. Using a value of 50,000 lipids perLiposome, the Liposome should have approximately 1000 maleimidemolecules per Liposome, about 600 of which are available on the outside.The Nucleic Acid Ligand-Liposome Complex was separated from the freeNucleic Acid Ligand via size exclusion chromatography (vide supra). Fromthe absorbance at 260 nm, it was estimated that approximately 200molecules of the Nucleic Acid Ligand were conjugated to each Liposome.

EXAMPLE 15

In Vitro and In Vivo Efficacy of Nucleic Acid Ligand-Liposome Complex,Dialkylglycerol (DAG)-Modified VEGF Ligand (NX278) Embedded in LiposomeBilayer.

NX278-Liposome Complex was prepared by incubating NX-278 (1 mg) (FIG.1N; SEQ ID NO: 19) with of a mixture of DSPC:cholesterol (50 mg) in 10mM phosphate (pH 7.4) buffer containing 9% sucrose and sonicated for15-30 min using a probe-type sonicator until opalescent solution wasobtained. The control Nucleic Acid Ligand-Liposome Complex containing asequence scrambled analog of ligand NX-278 (scNX278) (FIG. 1 W; SEQ IDNO:28) was prepared in the same manner. The size of Liposome particles(typically 50-100 nM), determined in a particle analyzer (Leeds &Northrup Model Microtrack UPA 150, Horsham, Pa.) was similar to thoseobtained in the absence of the Nucleic Acid Ligand. NX278-LiposomeComplex competed with a biotin-labeled Nucleic Acid Ligand to VEGF forbinding to polystyrene-immobilized VEGF in a competition ELISA assaywith an apparent ED50 of ≈10⁻⁷ M. In the same assay, scNX278-LiposomeComplex was not an effective competitor up to 2 μM Nucleic Acid Ligand.For comparison, free Nucleic Acid Ligand to VEGF with the same sequenceas NX278 but lacking the DAG moiety at the 5′ end, NX213 (FIG. 1P; SEQID NO:21), exhibited a competition ED50 value of ≈10⁻⁹ M. The reducedability of NX278-Liposome compared to NX213 to bind to immobilized VEGFmay be due to a simple geometric constraint, since only a fraction ofthe Nucleic Acid Ligand displayed on the outer surface of the Liposomesis expected to be available for binding to a planar surface. Inaddition, the fraction of Nucleic Acid Ligand displayed on the innersurface would obviously not be available for binding in this assay.

The effects of NX278-liposome, scNX278-liposome and NX213 on theproliferation of human umbilical vein endothelial cells (HUVEC) andKaposi's Sarcoma (KS) cells in tissue culture were examined. HUVECs weregrown in the presence of VEGF (10 ng/ml) in IMDM:Ham's F12 (1:1) mediumcontaining 10% fetal calf serum (FCS) and heparin (45 μg/ml). Cells wereplated in 24-well gelatin-coated plates at a density of 20,000 cells perwell on day zero and treated with the above ligands at concentrationsbetween 0.1 nM to 1 μM on days 1, 2, and 3 (replacing the media alongwith the ligands). Cell count was performed on day 4. KS cell line KSY-1was plated in 24-well gelatin coated plates at a density of 7,500-10,000cells per well on day zero in medium containing RPMI 1640 supplementedwith 2% FCS, L-glutamine, penicillin and streptomycin. Nucleic AcidLigands were added at concentrations between 0.1 nM to 1 μM in freshmedium on day 1, 2, and 3 and the cell count was performed on day 4.NX278-Liposome inhibited the proliferation of HUVECs with an IC50 of≈300 nM (the concentration refers to the Nucleic Acid Ligand component);the free Nucleic Acid analog, NX213, was significantly less effective(IC50>1 μM). NX278-Liposome also inhibited the proliferation of KS cellswith an IC50 of ≈100 nM; at 1 μM NX278-Liposome, the growth of thesecells was completely inhibited. scNX278-Liposome and NX213 exhibitedIC50 values of >1 μM.

The ability of NX278-liposome to inhibit the vascular permeabilityactivity of VEGF in vivo was examined. The vascular permeability assay(also known as the Miles assay (Miles, A. A. and Miles, E. M. (1952) J.Physiol. (London) 118:228) was performed in guinea pigs essentially asdescribed (Senger, R. S. et al., ( 1983) Science 219:983).NX278-Liposome at the concentration of 1 μM significantly inhibited theVEGF-induced vascular permeability increase. The control compound,scNX278-Liposome was not inhibitory at this concentration; in fact thevascular permeability appeared to be enhanced.

1. A therapeutic complex comprising a nucleic acid ligand and apolyethylene glycol (PEG) wherein said nucleic acid ligand is covalentlylinked to said PEG and wherein said nucleic acid ligand has a specificbinding affinity for vascular endothelial growth factor (VEGF), whereinsaid PEG is at least about 1000 Daltons.
 2. The complex of claim 1wherein said PEG is about 10,000 Daltons.
 3. The complex of claim 1wherein said PEG is about 20,000 Daltons.
 4. The complex of claim 1wherein said PEG is about 40,000 Daltons.
 5. The complex of claim 4wherein said nucleic acid ligand comprises a 2′-O methyl pyrimidinenucleotide.
 6. A method of improving the pharmacokinetic properties of anucleic acid ligand comprising covalently linking said nucleic acidligand to a PEG, wherein said nucleic acid ligand has a specific bindingaffinity for VEGF, wherein said PEG is at least about 1000 Daltons. 7.The method of claim 6 wherein said PEG is about 10,000 Daltons.
 8. Themethod of claim 6 wherein said PEG is about 20,000 Daltons.
 9. Themethod of claim 6 wherein said PEG is about 40,000 Daltons.
 10. Themethod of claim 9 wherein said nucleic acid ligand comprises a 2′-Omethyl pyrimidine nucleotide.